Stable formulations for cns delivery of arylsulfatase a

ABSTRACT

The present invention provides, among oilier things, compositions and methods for CNS delivery of lysosomal enzymes (e.g., recombinant human arylsulfatase A (rhASA)) for effective treatment of lysosomal storage diseases (e.g. Metachromatic Leukodystrophy Disease). In some embodiments, the present invention includes a stable formulation for intrathecal administration comprising an ASA protein and a poloxamer, wherein less than 3% of the ASA protein exists in aggregated form.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/580,064, filed Dec. 23, 2011, the entire contents of all of which areincorporated herein by reference.

BACKGROUND

Enzyme replacement therapy (ERT) involves the systemic administration ofnatural or recombinantly-derived proteins and/or enzymes to a subject.Approved therapies are typically administered to subjects intravenouslyand are generally effective in treating the somatic symptoms of theunderlying enzyme deficiency. As a result of the limited distribution ofthe intravenously administered protein and or enzyme into the cells andtissues of the central nervous system (CNS), the treatment of diseaseshaving a CNS etiology has been especially challenging because theintravenously administered proteins and or enzymes do not adequatelycross the blood-brain barrier (BBB).

The blood-brain barrier (BBB) is a structural system comprised ofendothelial cells that functions to protect the central nervous system(CNS) from deleterious substances in the blood, stream, such asbacteria, macromolecules (e.g., proteins) and other hydrophilic,molecules, by limiting the diffusion of such substances across the BBBand into the underlying cerebrospinal fluid (CSF) and CNS.

There are several ways of circumventing the BBB to enhance braindelivery of a therapeutic agent including direct intra-cranialinjection, transient permeabilization of the BBB, and modification ofthe active agent to alter tissue distribution. Direct injection of atherapeutic anent into brain tissue bypasses the vasculature completely,but suffers primarily from the risk of complications (infection, tissuedamage, immune responsive) incurred by intra-cranial injections and poordiffusion of the active agent from the site of administration. To date,direct administration of proteins into the brain substance has notachieved significant therapeutic effect due to diffusion barriers andthe limited volume of therapeutic that can be administered.Convection-assisted diffusion has been studied via catheters placed inthe brain parenchyma using slow, long-term infusions (Bobo, et al.,Proc. Natl. Acad. Set. USA 91, 2076-2080 (1994); Nguyen, et al, J.Neurosurg. 98, 584-590 (2003)), but no approved therapies currently usethis approach for long-term therapy in addition, the placement ofintracerebral catheters is very invasive and less desirable as aclinical alternative.

Intrathecal (IT) injection, or the administration of proteins to thecerebrospinal fluid (CSF), has also been attempted but has not yetyielded therapeutic success. A major challenge in this treatment hasbeen the tendency of the active agent to bind the ependymal lining ofthe ventricle very tightly which prevented subsequent diffusion.Currently, there are no approved products for the treatment of braingenetic disease by administration directly to the CSF.

In fact, many believed that the barrier to diffusion at the brainssurface, as well as the lack of effective and convenient deliverymethods, were too great an obstacle to achieve adequate therapeuticeffect in the brain for any disease.

Many lysosomal storage disorders affect the nervous system and thusdemonstrate unique challenges in treating these diseases withtraditional therapies. There is often a large build-up ofglycosaminoglycans (GAGS) in neurons and meninges of affectedindividuals, leading to various forms of CNS symptoms. To date, no CNSsymptoms resulting from a lysosomal disorder has successfully beentreated by any means available.

Furthermore, because proteins utilized in enzyme replacement therapy arelarger and more complex than traditional organic and inorganic drugs(i.e., possessing multiple functional groups in addition to complexthree-dimensional structures), the formulation, packaging andpreservation of such proteins poses special problems.

Thus, there remains a great need for formulations that can be used tosafely and effectively deliver therapeutic agents, such as lysosomalenzymes, to the brain.

SUMMARY OF THE INVENTION

The present invention provides improved formulations for intrathecaldelivery of lysosomal enzymes (e.g., arylsulfatase A (ASA)) for thetreatment of lysosomal storage diseases (e.g., MLD). As described in theExample section, the present invention is, in part, based on thesurprising discovery that poloxamer is particularly effective insuppressing enzyme precipitation/aggregation, under thermal andmechanical stress, including particulate formation such as flakes and/orfibers in saline or buffer based formulations. It is contemplated thatenzyme precipitation is in part caused by protein aggregation that formshigh molecular weight species (HMS), which are typically insoluble anddenatured. Protein aggregation is a common issue encountered duringmanufacture and other bioprocessing. For protein therapeutics, thepresence of aggregates of an type is typically considered to beundesirable because of the concern that the aggregates may lead to animmunogenic reaction (small aggregates) or may cause adverse events onadministration (particulates). The present invention encompasses therecognition that formulations containing a poloxamer stabilize lysosomalenzymes (e.g., ASA) and reduce protein aggregation duringlyophilization, reconstitution, storage and patient infusion. Thus,stable formulations according to the present invention can be used toeffectively deliver lysosomal enzymes such as an ASA protein,intrathecally for more effective treatment of lysosomal storage diseasesthat have CNS components without causing substantial immunogenicreaction and adverse effects. The invention further encompasses therecognition that use of a poloxamer allows for stable, ready-to-useliquid formulations, which can be stored at various temperatures.

As described in detail below, the present inventors have successfullydeveloped stable formulations for effective intrathecal (IT)administration of an arylsulfatase A (ASA) protein. It is contemplated,however, that various stable formulations described herein are generallysuitable for CNS delivery of therapeutic agents, including various otherlysosomal enzymes. Indeed, stable formulations according to the presentinvention can be used for CNS delivery via various techniques and routesincluding, but not limited to, intraparenchymal, intracerebral,intraventricular cerebral (ICV), intrathecal (e.g., IT-Lumbar.IT-cisterna magna) administrations and any other techniques and routesfor injection directly or indirectly to the CNS and/or CSF.

It is also contemplated that various stable formulations describedherein are generally suitable for CNS delivery of other therapeuticagents, such as therapeutic proteins including various replacementenzymes for lysosomal storage diseases. In some embodiments, areplacement enzyme can be a synthetic, recombinant, gene-activated ornatural enzyme.

In various aspects, the present invention includes a stable formulationair intrathecal administration comprising an arylsulfatase A (ASA)protein and a poloxamer, wherein less than about 5% (e.g., less thanabout 4%, less than about 3%, less than about 2%, less than about 1%,less than about 0.5%, less than about 0.1%) of the arylsulfatase A (ASA)protein exists in aggregated form. In some embodiments, the poloxamer isselected, from the croup consisting of Poloxamers 101, 105, 108, 122,123, 124, 181, 182, 183, 184, 185, 188, 212, 215, 217, 231, 234, 235,237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, 403, 407,and combination thereof. In certain embodiments, the poloxamer ispoloxamer 188.

In some embodiments, the poloxamer is present at a concentration ofapproximately 0.15%. In some embodiments, the poloxamer is present at aconcentration of approximately 0.1%. In certain embodiments, thepoloxamer is present at a concentration of approximately 0.05-0.5%,0.05-0.4%, 0.05-0.3%, 0.05%-0.2%, 0.05%-0.15%, 0.1-0.5%, 0.1-0.4%,0.1-0.3%, 0.1%-0.2%, or 0.1%-0.15%.

In certain embodiments, less than about 4% of the arylsulfatase A (ASA)protein exists in aggregated form. In certain embodiments, less thanabout 3% of the arylsulfatase A (ASA) protein exists in aggregated form.In certain embodiments, less than about 2% of the arylsulfatase A (ASA)protein exists in aggregated form. In certain embodiments, less thanabout 1% of the arylsulfatase A (ASA) protein exists in aggregated form.In certain embodiments, less than about 0.5% of the arylsulfatase A(ASA) protein exists in aggregated form.

In some embodiments, the ASA protein is present at a concentrationranging from approximately 0.1-100 mg/ml (e.g., approximately 1-100mg/ml, 5-100 mg/ml, 5-90 mg/ml, 5-80 mg/ml, 5-70 mg/ml, 5-60 mg/mL, 5-50mg/ml, 5-40mg/ml, 5-30 mg/ml, 5-20 mg/ml). In some embodiments, the ASAprotein is present at or greater than a concentration selected fromabout 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 10mg/ml, 15 mg/ml, 20mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 60mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, or 100 mg/ml. In certainembodiments, the ASA protein is present at a concentration selected fromabout 1 mg/ml, 10 mg/ml, 20 mg/ml, 30mg/ml, 50 mg/ml, or 100 mg/ml.

In some embodiments, the ASA protein comprises an amino acid sequencehaving at least about 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 1. In someembodiments, the ASA protein comprises an amino acid sequence of SEQ IDNO: 1.

In various embodiments, the ASA protein is produced from a human cellline. In certain embodiments, the ASA protein is produced from CHOcells.

In various embodiments the formulation further comprises salt. In someembodiments, the salt is NaCl. In certain embodiments, the NaCl ispresent as a concentration ranging from approximately 0-300 mM (e.g.,0-50 mM, 0-100 mM, 0-12.0 mM, 0-140 mM, 0-150 mM, 0-160 mM, 0-170 mM,0-180 mM, 0-200 mM, 0-250 mM). In certain embodiments, the NaCl ispresent at a concentration ranging from approximately 50-300 mM,approximately 50-200 mM, approximately 80-160 mM, approximately 100-160mM, approximately 110-180 mM, approximately 130-170 mM, or approximately1.30-160 mM. In certain embodiments, the NaCl is present at aconcentration of approximately 0 mM, 60 mM, 70 mM, 80 mM, 88 mM, 90 mM,100 mM, 110 mM, 120 mM, 130 mM, 132 mM, 137 mM, 138 mM, 140 mM, 142 mM,144 mM, 146 mM, 148 mM, 150 mM, 152 mM, 154 mM, 156 mM, 158 mM, or 160mM.

In various embodiments, the stable formulation further comprises abuffering agent. In certain embodiments, the buffering agent is selectedfrom the group consisting of phosphate, acetate, histidine, succinate,citrate, Tris, and combinations thereof. In some embodiments, thebuffering agent is phosphate. In some embodiments, the formulation has apH of approximately 3-8.0 (e.g., approximately 4-7.5, 5-8, 5-7.5, 5-6.5,5-7.0, 5.5-8.0, 5.5-7.7, 5.5-6.5, 6-7.5, or 6-7.0). In some embodiments,the formulation has a pH of approximately 6.0-6.5 (e.g., 6.0, 6.1, 6.2,6.3, 6.4, or 6.5). In certain embodiments, the formulation has a pH ofapproximately 6.0. In some embodiments, a stable formulation accordingto the invention does not comprise a buffering agent. As used herein, aformulation without a buffering agent is also known as a saline basedformulation.

In various embodiments, the formulation is a liquid formulation. Invarious embodiments, the formulation is formulated as lyophilized drypowder.

In various embodiments, the formulation further comprises a stabilizingagent. In certain embodiments, the stabilizing agent is selected fromthe group consisting of sucrose, glucose, mannitol, sorbitol,polyethylene glycol PEG2000, PEG3250, PEG4000, PEG5000, etc.),histidine, arginine, lysine, phospholipids, trehalose and combinationthereof.

In some embodiments, the present invention provides a stable formulationfor intrathecal administration comprising an arylsulfatase A (ASA)protein and a poloxamer, wherein the ASA protein is present at aconcentration of at least approximately 1 mg/ml (e.g., at leastapproximately 5 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, mg/ml, 40mg/ml, 45 mg/ml, 50 mg/ml). In some embodiments, the poloxamer is at aconcentration of approximately 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, or0.30%. In certain embodiments, the poloxamer is present at aconcentration of 0.05%-0.5%, 0.05%-0.2%, 0.05%-0.15%, 0.1%-0.2%, or0.1%-0.15%. In some embodiments, the poloxamer is selected from thegroup consisting of Poloxamers 101, 105, 108, 122, 123, 124, 181, 182,183, 184, 185, 188, 212, 215, 217, 231, 234, 235, 237, 238, 282, 284,288, 331, 333, 334, 335, 338, 401, 402, 403, 407, and combinationthereof. In certain embodiments, the poloxamer is poloxamer 188.

In some embodiments, less than about 5% (e.g., less than about 5%, lessthan about 4%, less than about 3%, less than about 2%, less than about1%, less than about 0.5%, or less than about 0.1%) of the arylsulfataseA (ASA) protein exists in aggregated form. In some embodiments, lessthan 3% of the arylsulfatase A (ASA) protein exists in aggregated form.In some embodiments, less than about 1% of the arylsulfatase A (ASA)protein exists in aggregated form.

In various embodiments, the ASA protein is at a concentration rangingfrom approximately 0.1-100 mg/ml. In some embodiments, the ASA proteinis present at or greater than a concentration selected from about 1mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml,25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 60 mg/ml, 70mg/ml, 80 mg/ml, 90 mg/ml, or 100 mg/ml.

In various aspects, the present invention includes a containercomprising a single dosage form of a stable formulation according toembodiments described herein. In some embodiments, the container isselected from an ampule, a vial, a cartridge, a reservoir, a lyo-ject,or a pre-filled syringe. In certain embodiments, the container is apre-filled syringe. In particular embodiments, the pre-filled syringe isselected from borosilicate glass syringes with baked silicone coating,borosilicate glass syringes with sprayed silicone, or plastic resinsyringes without silicone.

In some embodiments, the stable formulation is present in a volume ofless than about 50.0 mL (e.g., less than about 45 ml, 40 ml, 35 ml, 30ml, 25 ml, 20 ml, 15 ml, 10 ml, 5 ml, 4 ml, 3 ml, 2.5 ml, 2.0 ml, 1.5ml, 1.0 ml, or 0.5 ml). In certain embodiments, the stable formulationis present in a volume of less than about 5.0 mL (e.g., less than about5 ml, 4 ml, 2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml).

In various aspects, the present invention includes a method of treatingmetachromatic leukodystrophy (MLD) disease comprising a step ofadministering intrathecally to a subject in need of treatment aformulation according to any of the embodiments described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are thr illustration purposes only and not for limitation,

FIG. 1 illustrates exemplary arylsulfatase A (rhASA) formulationagitation studies.

FIGS. 2 and 3 illustrate exemplary rhASA pumping and shaking studies.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Amelioration: As used herein, the term “amelioration” is meant theprevention, reduction or palliation of a state, or improvement of thestate of a subject. Amelioration includes, but does not require completerecovery or complete prevention of a disease condition. In someembodiments, amelioration includes increasing levels of relevant proteinor its activity that is deficient in relevant disease tissues.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active. In particularembodiments, where a protein or polypeptide is biologically active, aportion of that protein or polypeptide that shares at least onebiological activity of the protein or polypeptide is typically referredto as a “biologically active” portion.

Bulking agent: As used herein, the term “bulking agent” refers to acompound which adds mass to the lyophilized mixture and contributes tothe physical structure of the lyophilized cake (e.g., facilitates theproduction of an essentially uniform lyophilized cake which maintains anopen pore structure). Exemplary bulking agents include mannitol,glycine, sodium chloride, hydroxyethyl starch, lactose, sucrose,trehalose, polyethylene glycol and dextran.

Cation-independent mannose-6-phosphate receptor (CI-MPR): As usedherein, the term “cation-independent mannose-6-phosphate receptor(CI-MPR)” refers to a cellular receptor that binds mannose-6-phosphate(M6P) tags on acid hydrolase precursors in the Golgi apparatus that aredestined for transport to the lysosome. In addition tomannose-6-phosphates, the CI/MPR also hinds other proteins includingIGF-II. The CI-MPR is also known as “M6P/IGF-II receptor,”“CI-MPR/IGF-II receptor,” “IGF-II receptor” or “IGF2 Receptor.” Theseterms and abbreviations thereof are used interchangeably herein.

Concurrent immunosuppressant therapy: As used herein, the term“concurrent immunosuppressant therapy” includes any immunosuppressanttherapy used as pre-treatment, preconditioning or in parallel to atreatment method.

Diluent: As used herein, the term “diluent” refers to a pharmaceuticallyacceptable (e.g., safe and non-toxic for administration to at human)diluting substance useful for the preparation of at reconstitutedformulation. Exemplary diluents include sterile water, bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

Dosage form: As used herein, the terms “dosage form” and “unit dosageform” refer to a physically discrete unit of a therapeutic protein forthe patient to be treated. Each unit contains a predetermined quantityof active material calculated to produce the desired therapeutic effect.It will be understood, however, that the total dosage of the compositionwill be decided by the attending physician within the scope of soundmedical judgment.

Enzyme replacement they (ERT): As used herein, the term “enzymereplacement therapy (ERT)” refers to any therapeutic strategy thatcorrects an enzyme deficiency by providing the missing enzyme. In someembodiments, the missing enzyme is provided, by intrathecaladministration. In some embodiments, the missing enzyme is provided byinfusing into bloodstream. Once administered, enzyme is taken up bycells and transported to the lysosome, where the enzyme acts toeliminate material that has accumulated in the lysosomes due to theenzyme deficiency. Typically, for lysosomal enzyme replacement therapyto be effective, the therapeutic enzyme is delivered to lysosomes in theappropriate cells in target tissues where the storage defect ismanifest.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein. A“control individual” is an individual afflicted with the same form oflysosomal storage disease as the individual being treated, who is aboutthe same age as the individual being treated (to ensure that the stagesof the disease in the treated individual and the control individual(s)are comparable).

Individual, subject, patient: As used herein, the terms “subject,”“individual” or “patient” refer to a human or a non-human mammaliansubject. The individual (also referred to as “patient” or “subject”)being treated is an individual (fetus, infant, child, adolescent, oradult human) suffering from a disease.

Intrathecal administration: As used herein, the term “intrathecaladministration” or “intrathecal injection” refers to an injection intothe spinal canal (intrathecal space surrounding the spinal cord).Various techniques may be used including, without limitation, lateralcerebroventricular injection through a burrhole or cisternal or lumbarpuncture or the like. In some embodiments, “intrathecal administration”or “intrathecal delivery” according to the present invention refers toIT administration or delivery via the lumbar area or region, i.e.,lumbar IT administration or delivery. As used herein, the term “lumbarregion” or “lumbar area” refers to the area between the third and fourthlumbar (lower back) vertebrae and, more inclusively the L2-S1 region ofthe spine.

Linker: As used herein, the term “linker” refers to, in a fusionprotein, an amino acid sequence other than that appearing at aparticular position in the natural protein and is generally designed tobe flexible or to interpose a structure, such as an a-helix, between twoprotein moieties. A linker is also referred to as a spacer.

Lyoprotectant: As used herein, the term “lyoprotectant” refers to amolecule that prevents or reduces chemical and/or physical instabilityof a protein or other substance upon lyophilization and subsequentstorage. Exemplary lyoprotectants include sugars such as sucrose ortrehalose: an amino acid such as monosodium glutamate or histidine; amethylamine such as betaine; a lyotropic salt such as magnesium sulfate;a polyol such as trihydric or higher sugar alcohols, e.g. glycerin,erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol;propylene glycol; polyethylene glycol; Pluronics; and combinationsthereof. In some embodiments, a lyoprotectant is a non-reducing sugar,such as trehalose or sucrose.

Lysosomal enzyme: As used herein, the term “lysosomal enzyme” refers toany enzyme that is capable of reducing accumulated materials inmammalian lysosomes or that can rescue or ameliorate one or morelysosomal storage disease symptoms. Lysosomal enzymes suitable for theinvention include both wild-type or modified lysosomal enzymes and canbe produced using recombinant and synthetic methods or purified fromnature sources. Exemplary lysosomal enzymes are listed in Table 1.

Lysosomal enzyme deficiency: As used herein, “lysosomal enzymedeficiency” refers to a group of genetic disorders that result fromdeficiency in at least one of the enzymes that are required to breakmacromolecules (e.g., enzyme substrates) down to peptides, amino acids,monosaccharides, nucleic acids and fatty acids in lysosomes. As aresult, individuals suffering from lysosomal enzyme deficiencies haveaccumulated materials in various tissues (e.g., CNS, liver, spleen, gut,blood vessel walls and other organs).

Lysosomal Storage Disease: As used herein, the term “lysosomal storagedisease” refers to any disease resulting from the deficiency of one ormore lysosomal enzymes necessary for metabolizing naturalmacromolecules. These diseases typically result in the accumulation ofun-degraded molecules in the lysosomes, resulting in increased numbersof storage granules (also termed storage vesicles). These diseases andvarious examples are described in more detail below.

Polypeptide: As used herein, a “polypeptide”, generally speaking, is astring of at least two amino acids attached to one another by a peptidebond. In some embodiments, a polypeptide may include at least 3-5 aminoacids, each of which is attached to others by way of at least onepeptide bond. Those of ordinary skill in the art will appreciate thatpolypeptides sometimes include “non-natural” amino acids or otherentities that nonetheless are capable of integrating into as polypeptidechain, optionally.

Replacement enzyme: As used herein, the term “replacement enzyme” refersto any enzyme that can act to replace at least in part the deficient ormissing enzyme in a disease to be treated. In some embodiments, the term“replacement enzyme” refers to any enzyme that can act to replace atleast in part the deficient or missing lysosomal enzyme in a lysosomalstorage disease to be treated. In some embodiments, a replacement enzymeis capable of reducing accumulated materials in mammalian lysosomes orthat can rescue or ameliorate one or more lysosomal storage diseasesymptoms. Replacement enzymes suitable for the invention include bothwild-type or modified lysosomal enzymes and can be produced usingrecombinant and synthetic methods or purified from nature sources. Areplacement enzyme can be a recombinant, synthetic, gene-activated ornatural enzyme.

Soluble: As used herein, the term “soluble” refers to the ability of atherapeutic agent to form a homogenous solution. In some embodiments,the solubility of the therapeutic agent in the solution into which it isadministered and by which it is transported to the target site of action(e.g., the cells and tissues of the brain) is sufficient to permit thedelivery of a therapeutically effective amount of the therapeutic agentto the targeted, site of action. Several factors can impact thesolubility of the therapeutic agents. For example, relevant factorswhich may impact protein solubility include ionic strength, amino acidsequence and the presence of other co-solubilizing agents or salts(e.g., calcium salts). In some embodiments, the pharmaceuticalcompositions are formulated such that calcium salts are excluded fromsuch compositions. In some embodiments, therapeutic agents in accordancewith the present invention are soluble in its correspondingpharmaceutical composition. It will be appreciated that, while isotonicsolutions are generally preferred for parenterally administered drugs,the use of isotonic solutions may limit adequate solubility for sometherapeutic agents and, in particular some proteins and/or enzymes.Slightly hypertonic solutions (e.g., up to 175 mM sodium chloride in 5mM sodium phosphate at pH 7.0) and sugar-containing solutions (e.g., upto 2% sucrose in 5 mM sodium phosphate at pH 7.0) have been demonstratedto be well tolerated in monkeys. For example, the most common approvedCNS bolus formulation composition is saline (150 mM NaCl in water).

Stability: As used herein, the term “stable” refers to the ability ofthe therapeutic agent (e.g., a recombinant enzyme) to maintain itstherapeutic efficacy (e.g., all or the majority of its intendedbiological activity and/or physiochemical integrity) over extendedperiods of time. The stability of a therapeutic agent, and thecapability of the pharmaceutical composition to maintain stability ofsuch therapeutic agent, may be assessed over extended periods of time(e.g., for at least 1, 3, 6, 12, 18, 24, 30, 36 months or more). Ingeneral, pharmaceutical compositions described herein have beenformulated such that they are capable of stabilizing, or alternativelyslowing or preventing the degradation, of one or more therapeutic agentsformulated therewith (e.g., recombinant proteins). In the context of aformulation a stable formulation is one in which the therapeutic agenttherein essentially retains us physical and/or chemical integrity andbiological activity upon storage and during processes (such asfreeze/thaw, mechanical mixing and lyophilization). For proteinstability, it can be measure by formation of high molecular weight (HMW)aggregates, loss of enzyme activity, generation of peptide fragments andshill of charge profiles.

Subject: As used herein, the term “subject” means any mammal, includinghumans. In certain embodiments of the present invention the subject isan adult, an adolescent or an infant. Also contemplated by the presentinvention are the administration of the pharmaceutical compositionsand/or performance of the methods of treatment in-utero.

Substantial homology: The phrase “substantial homology” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially homologous” ifthey contain homologous residues in corresponding positions. Homologousresidues may be identical residues. Alternatively, homologous residuesmay be non-identical residues will appropriately similar structuraland/or functional characteristics. For example, as is well known bythose of ordinary skill in the art, certain amino acids are typicallyclassified as “hydrophobic” or “hydrophilic” amino acids, and/or ashaving “polar” or “non-polar” side chains Substitution of one amino acidfor another of the same type may often be considered a “homologous”substitution.

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol. 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.In addition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91% 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 100,325, 350, 375, 400, 425, 450, 475, 500 or more residues.

Substantial identity: The Phrase “substantial identity” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially identical” ifthey contain identical residues in corresponding positions. As is wellknown in this art, amino acid or nucleic acid sequences may be comparedusing any of a variety of algorithms, including those available incommercial computer programs such as BLASTN for nucleotide sequences andBLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplarysuch programs are described in Altschul, et al., Basic local alignmentsearch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al.,Methods Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402,1997; Baxevanis et al., Bioinformatics: A Practical Guide to theAnalysis of Genes and Proteins, Wiley, 1998; and Misener, et al.,(eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132), Humana Press, 1999, in addition to identifyingidentical sequences, the programs mentioned above typically provide anindication of the degree of identity. In some embodiments, two sequencesare considered to be substantially identical if at least 50%, 55%, 60%,65%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore of their corresponding residues are identical over a relevantstretch of residues. In some embodiments, relevant stretch is a completesequence. In some embodiments, the relevant stretch is at least 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500 or more residues.

Synthetic CSF: As used herein, the term “synthetic CSF” refers to asolution that has pH, electrolyte composition, glucose content andosmalarity consistent with the cerebrospinal fluid. Synthetic CSF isalso referred to as artificial CSF. In some embodiments, synthetic CSFis an Elliott's B solution.

Suitable for CNS delivery: A used herein, the phrase “suitable for CNSdelivery” or “suitable for intrathecal delivery” as it relates to thepharmaceutical compositions of the present invention generally refers tothe stability, tolerability, and solubility properties of suchcompositions, as well as the ability of such compositions to deliver aneffective amount of the therapeutic agent contained therein to thetargeted site of delivery (e.g., the CSF or the brain).

Target tissues: As used herein, the term “target tissues” refers to anytissue that is affected by the lysosomal storage disease to be treatedor any tissue in which the deficient lysosomal enzyme is normallyexpressed. In some embodiments, target tissues include those tissues inwhich there is a detectable or abnormally high amount of enzymesubstrate, for example stored in the cellular lysosomes of the tissue,in patients suffering from or susceptible to the lysosomal storagedisease. In some embodiments, target tissues include those tissues thatdisplay disease-associated pathology, symptom, or feature. In someembodiments, target tissues include those tissues in which the deficientlysosomal enzyme is normally expressed at an elevated level. As usedherein, a target tissue may be a brain target tissue, a spinal cordtarget tissue and/or a peripheral target tissue. Exemplary targettissues are described in detail below.

Therapeutic moiety: As used herein, the term “therapeutic moiety” refersto a portion of a molecule that renders the therapeutic effect of themolecule. In some embodiments, a therapeutic moiety is a polypeptidehaving therapeutic activity.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount of a therapeuticprotein (e.g., replacement enzyme) which confers a therapeutic effect onthe treated subject, at a reasonable benefit/risk ratio applicable toany medical treatment. The therapeutic effect may be objectivemeasurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). In particular, the “therapeuticallyeffective amount” refers to an amount of a therapeutic protein orcomposition effective to treat, ameliorate, or prevent a desired diseaseor condition, or to exhibit a detectable therapeutic or preventativeeffect, such as by ameliorating symptoms associated with the disease,preventing or delaying the onset of the disease, and/or also lesseningthe severity or frequency of symptoms of the disease. A therapeuticallyeffective amount is commonly administered in a dosing regimen that maycomprise multiple unit doses. For any particular therapeutic protein, astherapeutically effective amount (and/or an appropriate unit dose withinan effective dosing regimen) may vary, for example, depending on routeof administration, on combination with other pharmaceutical agents.Also, the specific therapeutically effective amount (and/or unit dose)for any particular patient may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific pharmaceutical agent employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration,and/or rate of excretion or metabolism of the specific fusion proteinemployed; the duration of the treatment; and like factors as is wellknown in the medical arts.

Tolerable: As used herein, the terms “tolerable” and “tolerability”refer to the ability of the pharmaceutical compositions of the presentinvention to not elicit an adverse reaction in the subject to whom suchcomposition is administered, or alternatively not to elicit a seriousadverse reaction in the subject to whom such composition isadministered. In some embodiments, the pharmaceutical compositions ofthe present invention are well tolerated by the subject to whom suchcompositions is administered.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a therapeutic proteinlysosomal enzyme) that partially or completely alleviates, ameliorates,relieves, inhibits, delays onset of, reduces severity of and/or reducesincidence of one or more symptoms or features of a particular disease,disorder, and/or condition (e.g., Hunter syndrome, Sanfilippo Bsyndrome). Such treatment may be of a subject who does not exhibit signsoldie relevant disease, disorder and/or condition and/or of a subjectwho exhibits only early signs of the disease, disorder, and/orcondition. Alternatively or additionally, such treatment may be of asubject who exhibits one or more established signs of the relevantdisease, disorder and/or condition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, among other things, improved methods andcompositions for effective direct delivery of a therapeutic agent to thecentral nervous system (CNS). The present invention is, in pan, based onthe surprising discovery that the including surfactant, in particular, apoloxamer such as poloxamer 188, in a formulation significantly reducedor eliminated enzyme precipitation. As discussed above, enzymeprecipitation is in part caused by protein aggregation that forms highmolecular weight species (HMS), which are typically insoluble anddenatured. Protein aggregation is a common issue encountered duringmanufacture and other bioprocessing (e.g., lyophilization,reconstitution, storage and patient infusion). For protein therapeutics,the presence of aggregates of any type is typically considered to beundesirable because of the concern that the aggregates may lead to animmunogenic reaction (small aggregates) or may cause adverse events onadministration (particulates). Thus, stable formulations according tothe present invention can be used to effectively deliver lysosomalenzymes, such as an ASA protein, intrathecally for more effectivetreatment of lysosomal storage diseases that have CNS components withoutcausing substantial immunogenic reaction and adverse effects.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention in thisapplication, the use of “or” means “and/or” unless stated otherwise.

Therapeutic Proteins

In some embodiments, inventive methods and compositions provided by thepresent invention are used to deliver an arylsulfatase A (ASA) proteinto the CNS for treatment of Metachromatic Leukodystrophy Disease. Asuitable ASA protein can be any molecule or as portion of a moleculethat can substitute for naturally-occurring arylsulfatase A (ASA)protein activity or rescue one or more phenotypes or symptoms associatedwith ASA-deficiency. In some embodiments, as replacement enzyme suitablefor the invention is a polypeptide having an N-terminus and a C-terminusand an amino acid sequence substantially similar or identical to maturehuman ASA protein.

Typically, human ASA is produced as a precursor molecule that isprocessed to a mature form. This process generally occurs by removingthe 18 amino acid signal peptide. Typically, the precursor form is alsoreferred to as full-length precursor or full-length ASA protein, whichcontains 507 amino acids. The N-terminal 18 amino acids are cleaved,resulting in a mature form that is 489 amino acids in length. Thus, itis contemplated that the N-terminal 18 amino acids is generally notrequired for the ASA protein activity. The amino acid sequences of themature form (SEQ ID NO:1) and lull-length precursor (SEQ ID NO:2) of atypical wild-type or naturally-occurring human ASA protein are shown inTable 1.

TABLE 1 Human Arylsulfatase A Mature FormRPPNIVLIFADDLGYGDLGCYGHPSSTTPNLDQLA AGGLRFTDFYVPVSLCTPSRAALLTGRLPVRMGMYPGVLVPSSRGGLPLEEVTVAEVLAARGYLTGMAGK WHLGVGPEGAFLPPHQGFHRFLGIPYSHDQGPCQMLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPW LPGLEARYMAFAHDLMADAQRQDRPFFLYYASHHTHYPQFSGQSFAERSGRGPFGDSLMELDAAVGTLMT AIGDLGLLEETLVIFTADNGPETMRMSRGGCSGLLRCGKGTTYEGGVREPALAFWPGHIAPGVTHELASS LDLLPTLAALAGAPLPNVTLDGFDLSPLLLGTGKSPRQSLFFYPSYPDEVRGVFAVRTGKYKAHFFTQGS AHSDTTADPACHASSSLTAHEPPLLYDLSKDPGENYNLLGGVAGATPEVLQALKQLQLLKAQLDAAVTFG PSQVARGEDPALQICCHPGCTPRPACCHCPDPHA(SEQ ID NO: 1) Full-Length MGAPRSLLLALAAGLAVARPPNIVLIFADDLGYGD PrecursorLGCYGHPSSTTPNLDQLAAGGLRFTDFYVPVSLCT PSRAALLTGRLPVRMGMYPGVLVPSSRGGLPLEEVTVAEVLAARGYLTGMAGKWHLGVGPEGAFLPPHQG FHRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEARYMAFAHDLMA DAQRQDRPFFLYYASHHTHYPQFSGQSFAERSGRGPFGDSLMELDAAVGTLMTAIGDLGLLEETLVIFTA DNGPETMRMSRGGCSGLLRCGKGTTYEGGVREPALAPWPGHIAPGVTHELASSLDLLPTLAALAGAPLPN VTLDGFDLSPLLLGTGKSPRQSLFFYPSYPDEVRGVFAVRTGKYKAHFFTQGSAHSDTTADPACHASSSL TAHEPPLLYDLSKDPGENYNLLGGVAGATPEVLQALKQLQLLKAQLDAAVTFGPSQVARGEDPALQICCH PGCTPRPACCHCPDPHA (SEQ ID NO: 2)

Thus, in some embodiments, a therapeutic moiety suitable for the presentinvention is mature human ASA protein (SEQ ID NO:1). In someembodiments, a suitable therapeutic moiety may be a homologue or ananalogue of mature human ASA protein. For example, as homologue or ananalogue of mature human ASA protein may be as modified mature human ASAprotein containing one or more amino acid substitutions, deletions,and/or insertions as compared to a wild-type or naturally-occurring ASAprotein (e.g., SEQ ID NO:1), while retaining substantial ASA proteinactivity. Thus, in some embodiments, a therapeutic moiety suitable forthe present invention is substantially homologous to mature human ASAprotein (SEQ ID NO:1). In some embodiments, a therapeutic moietysuitable for the present invention has an amino acid sequence at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more homologous to SEQ ID NO:1. In someembodiments, a therapeutic moiety suitable for the present invention issubstantially identical to mature human ASA protein (SEQ ID NO:1). Insome embodiments, a therapeutic moiety suitable for the presentinvention has an amino acid sequence at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to SEQ ID NO:1. In some embodiments, a therapeutic moietysuitable for the present invention contains a fragment or a portion ofmature human ASA protein.

Alternatively, a replacement enzyme suitable for the present inventionis full-length ASA protein. In some embodiments, a suitable replacementenzyme may be a homologue or an analogue of full-length human ASAprotein. For example, a homologue or an analogue of full-length humanASA protein may be a modified foil-length human ASA protein containingone or more amino acid substitutions, deletions, and/or insertions ascompared to a wild-type or naturally-occurring full-length ASA protein(e.g., SEQ ID NO:2), while retaining substantial ASA protein activity.Thus, in some embodiments, a replacement enzyme suitable for the presentinvention is substantially homologous to full-knob human ASA protein(SEQ ID NO:2). In some embodiments, at replacement enzyme suitable forthe present invention has an amino acid sequence at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 97%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more homologous to SEQ ID NO:2. In some embodiments, areplacement enzyme suitable for the present invention is substantiallyidentical to SEQ ID NO:2. In some embodiments, a replacement enzymesuitable for the present invention has an amino acid sequence at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to SEQ ID NO:2. In someembodiments, a replacement enzyme suitable for the present inventioncontains a fragment or a portion of full-length human ASA protein. Asused herein, a full-length ASA protein typically contains signal peptidesequence.

In some embodiments, a therapeutic protein includes a targeting moiety(e.g., a lysosome targeting sequence) and/or a membrane-penetratingpeptide. In some embodiments, a targeting sequence and/or amembrane-penetrating peptide is an intrinsic part of the therapeuticmoiety (e.g., via a chemical linkage, via a fusion protein). In someembodiments, a targeting sequence contains a mannose-6-phosphate moiety.In some embodiments, a targeting sequence contains an IGF-I moiety. Insome embodiments, a targeting sequence contains an moiety.

Other Lysosomal Storage Diseases and Replacement Enzymes

It is contemplated that inventive methods and compositions according tothe present invention can be used to treat other lysosomal storagediseases, in particular those lysosomal storage diseases having CNSetiology and/or symptoms, including, but are not limited to,aspartylglucosaminuria, cholesterol ester storage disease, Wolmandisease, cystinosis, Danon disease, Fabry disease, Farberlipogranulomatosis, Farber disease, fucosidosis, galactosialidosis typesI/II, Gaucher disease types I/II/III, globoid cell leukodystrophy,Krabbe disease, glycogen storage disease II, Pompe disease,GM1-gangliosidosis types I/II/III, GM2-gangliosidosis type I, Tay Sachsdisease, GM2-gangliosidosis type II, Sandhoff disease,GM2-gangliosidosis, α-mannosidosis types I/II, .beta.-mannosidosis,metachromatic leukodystrophy, mucolipidosis type I, sialidosis typesI/II, mucolipidosis types II/III, I-cell disease, mucolipidosis typeIIIC pseudo-Hurler polydystrophy, mucopolysaccharidosis type I,mucopolysaccharidosis type II, mucopolysaccharidosis type IIIA,Sanfilippo syndrome, mucopolysaccharidosis type IIIB,mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID,mucopolysaccharidosis type IVA, Morquio syndrome, mucopolysaccharidosistype IVB, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII,Sly syndrome, mucopolysaccharidosis type IX, multiple sulfatasedeficiency, neuronal ceroid lipofuscinosis, CLN1 Batten disease, CLN2Batten disease, Niemann-Pick disease types A/B, Niemann-Pick diseasetype C1, Niemann-Pick disease type C2, pycnodysostosis, Schindlerdisease types I/II, Gaucher disease and sialic acid storage disease.

A detailed review of the genetic etiology, clinical manifestations, andmolecular biology of the lysosomal storage diseases are detailed inScriver et al., eds., The Metabolic and Molecular Basis of InheritedDisease, 7.sup.th Ed., Vol. II, McGraw Hill, (1995). Thus, the enzymesdeficient in the above diseases are known to those of skill in the art,some of these are exemplified in Table 2 below:

TABLE 2 Substance Disease Name Enzyme Deficiency Stored Pompe DiseaseAcid-a1,4- Glycogen α□1-4 Glucosidase linked Oligosaccharides GM1Gangliodsidosis β-Galactosidase GM₁ Gangliosides Tay-Sachs Diseaseβ-Hexosaminidase A GM₂ Ganglioside GM2 Gangliosidosis: GM₂ Activator GM₂Ganglioside AB Variant Protein Sandhoff Disease β-Hexosaminidase GM₂Ganglioside A&B Fabry Disease α-Galactosidase A Globosides GaucherDisease Glucocerebrosidase Glucosylceramide Metachromatic ArylsulfataseA Sulphatides Leukodystrophy Krabbe Disease GalactosylceramidaseGalactocerebroside Niemann Pick, Types Acid Sphingomyelin A & BSphingomyelinase Niemann-Pick, Type C Cholesterol SphingomyelinEsterification Defect Niemann-Pick, Type D Unknown Sphingomyelin FarberDisease Acid Ceramidase Ceramide Wolman Disease Acid Lipase CholesterylEsters Hurler Syndrome α-L-Iduronidase Heparan & (MPS IH) DermatanSulfates Scheie Syndrome α-L-Iduronidase Heparan & (MPS IS) Dermatan,Sulfates Hurler-Scheie α-L-Iduronidase Heparan & (MPS IH/S) DermatanSulfates Hunter Syndrome Iduronate Sulfatase Heparan & (MPS II) DermatanSulfates Sanfilippo A Heparan N-Sulfatase Heparan (MPS IIIA) SulfateSanfilippo B α-N- Heparan (MPS IIIB) Acetylglucosaminidase SulfateSanfilippo C Acetyl-CoA- Heparan (MPS IIIC) Glucosaminide SulfateAcetyltransferase Sanfilippo D N-Acetylglucosamine- Heparan (MPS IIID)6-Sulfatase Sulfate Morquio B β-Galactosidase Keratan (MPS IVB) SulfateMaroteaux-Lamy Arylsulfatase B Dermatan (MPS VI) Sulfate Sly Syndromeβ-Glucuronidase (MPS VII) α-Mannosidosis α-Mannosidase Mannose/Oligosaccharides β-Mannosidosis β-Mannosidase Mannose/ OligosaccharidesFucosidosis α-L-Fucosidase Fucosyl OligosaccharidesAspartylglucosaminuria N-Aspartyl-β- Aspartylglucosamine GlucosaminidaseAsparagines Sialidosis α-Neuraminidase Sialyloligosaccharides(Mucolipidosis I) Galactosialidosis Lysosomal ProtectiveSialyloligosaccharides (Goldberg Syndrome) Protein Deficiency SchindlerDisease α-N-Acetyl- Galactosaminidase Mucolipidosis II (I-N-Acetylglucosamine- Heparan Sulfate Cell Disease) 1-PhosphotransferaseMucolipidosis III Same as ML II (Pseudo-Hurler Polydystrophy) CystinosisCystine Transport Free Cystine Protein Salla Disease Sialic AcidTransport Free Sialic Acid and Protein Glucuronic Acid Infantile SialicAcid Sialic Acid Transport Free Sialic Acid and Storage Disease ProteinGlucuronic Acid Infantile Neuronal Palmitoyl-Protein Lipofuscins CeroidLipofuscinosis Thioesterase Mucolipidosis IV Unknown Gangliosides &Hyaluronic Acid Prosaposin Saposins A, B, C or D

Inventive methods according to the present invention may be used todeliver various other replacement enzymes. As used herein, replacementenzymes suitable for the present invention may include any enzyme thatcan act to replace at least partial activity of the deficient or missinglysosomal enzyme in a lysosomal storage disease to be treated. In someembodiments, a replacement enzyme is capable of reducing accumulatedsubstance in lysosomes or that can rescue or ameliorate one or morelysosomal storage disease symptom.

In some embodiments, a suitable replacement enzyme may be any lysosomalenzyme known to be associated with the lysosomal storage disease to betreated. In some embodiments, a suitable replacement enzyme is an enzymeselected from the enzyme listed in Table 2 above.

In some embodiments, a replacement enzyme suitable for the invention mayhave a wild-type or naturally occurring sequence. In some embodiments, areplacement enzyme suitable for the invention may have a modifiedsequence having substantial homology or identify to the wild-type ornaturally-occurring sequence (e.g., having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity to the wild-type ornaturally-occurring sequence).

A replacement enzyme suitable for the present invention ma be producedby any available means. For example, replacement enzymes may berecombinantly produced by utilizing a host cell system engineered toexpress a replacement enzyme-encoding nucleic acid. Alternatively oradditionally, replacement enzymes may be produced by activatingendogenous genes. Alternatively or additionally, replacement enzymes maybe partially or fully prepared by chemical synthesis. Alternatively oradditionally, replacements enzymes may also be purified from naturalsources.

Where enzymes are recombinantly produced, any expression system can beused. To give but a few examples, known expression systems include, forexample, egg, baculovirus, plant, yeast, or mammalian cells.

In some embodiments, enzymes suitable for the present invention areproduced in mammalian cells. Non-limiting examples of mammalian cellsthat may be used in accordance with the present invention include BALB/cmouse myeloma line (NSO/1, ECACC No: 85110503); human retinoblasts(PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36:59, 1977); human fibrosarcoma cell line (e.g.,HT1080); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells +/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,774216, 1980); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76ATCC CRL-1 587); human cervical carcinomacells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

In some embodiments, inventive methods according to the presentinvention are used to deliver replacement enzymes produced from humancells. In some embodiments, inventive methods according to the presentinvention are used to deliver replacement enzymes produced from CHOcells.

In some embodiments, replacement enzymes delivered using a method of theinvention contain a moiety that binds to a receptor on the surface ofbrain cells to facilitate cellular uptake and/or lysosomal targeting.For example, such a receptor may be the cation, independentmannose-6-phosphate receptor (CI-MPR) which binds themannose-6-phosphate (M6P) residues. In addition, the CI-MPR also bindsother proteins including IGF-II. In some embodiments, a replacementenzyme suitable for the present invention contains M6P residues on thesurface of the protein. In some embodiments, a replacement enzymesuitable for the present invention ma contain bis-phosphorylatedoligosaccharides which have higher binding affinity to the CI-MPR. Insome embodiments, a suitable enzyme contains up to about an average ofabout at least 20% bis-phosphorylated oligosaccharides per enzyme. Inother embodiments, a suitable enzyme may contain about 10%, 15%, 18%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% bis-phosphorylatedoligosaccharides per enzyme. While such bis-phosphorylatedoligosaccharides may be naturally present on the enzyme, it should benoted that the enzymes may be modified to possess such oligosaccharides.For example, suitable replacement enzymes may be modified by certainenzymes which are capable of catalyzing the transfer ofN-acetylglucosamine-L-phosphate from UDP-GlcNAc to the 6′ position ofα-1,2-linked mannoses on lysosomal enzymes. Methods and compositions forproducing and using such enzymes are described by, for example, Canfieldet al. in U.S. Pat. No. 6,537,785, and U.S. Pat. No. 6,534,300, eachincorporated herein by reference.

In some embodiments, replacement enzymes for use in the presentinvention may be conjugated or fused to a lysosomal targeting moietythat is capable of binding to a receptor on the surface of brain cells.A suitable lysosomal targeting moiety can be IGF-II, RAP, p97, andvariants, homologues or fragments thereof (e.g., including those peptidehaving a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical toa wild-type mature human IGF-I, IGF-II, RAP, p97 peptide sequence).

In some embodiments replacement enzymes suitable for the presentinvention have not been modified to enhance delivery or transport ofsuch agents across the BBB and into the CNS.

In some embodiments, a therapeutic protein includes a targeting moiety(e.g., lysosome targeting sequence) and/or a membrane-penetratingpeptide. In some embodiments, a targeting sequence and/or amembrane-penetrating peptide is an intrinsic part of the therapeuticmoiety (e.g., via a chemical linkage, via a fusion protein). In someembodiments, a targeting sequence contains a mannose-6-phosphate moiety.In some embodiments, a targeting sequence contains an IGF-I moiety. Insome embodiments, a targeting sequence contains an IGF-II moiety.

Formulations

Aqueous pharmaceutical solutions and compositions (i.e., formulations)that are traditionally used to deliver therapeutic agents to the CNS ofa subject include unbuffered isotonic saline and Elliott's B solution,which is artificial CSF. A comparison depicting the compositions of CSFrelative to Elliott's B solution is included in Table 3 below. As shownin Table 3, the concentration of Elliot's B Solution closely parallelsthat of the CSF. Elliott's B Solution, however contains a very lowbuffer concentration and accordingly may not provide the adequatebuffering capacity needed to stabilize therapeutic agents (e.g.,proteins), especially over extended periods of time (e.g., duringstorage conditions). Furthermore Elliott's B Solution contains certainsalts which may be incompatible with the formulations intended todeliver some therapeutic agents, and in particular proteins or enzymes.For example, the calcium salts present in Elliott's B Solution arecapable of mediating protein precipitation and thereby reducing thestability of the formulation.

TABLE 3 Na⁺ K⁺ Ca⁺⁺ Mg⁺⁺ HCO3⁻ Cl⁻ Phosphorous Glucose Solution mEq/LmEq/L mEq/L mEq/L mEq/L mEq/L pH mg/L mg/L CSF 117-137 2.3 2.2 2.2 22.9113-127 7.31 1.2-2.1 45-80 Elliott's 149 2.6 2.7 2.4 22.6 132 6.0-7.52.3 80 B Sol'n

The present invention provides formulations, in either aqueous,pre-lyophilized, lyophilized or reconstituted form for therapeuticagents that have been formulated such that they are capable ofstabilizing, or alternatively slowing or preventing the degradation, ofone or more therapeutic agents form dated therewith (e.g., recombinantproteins). In some embodiments, the present formulations providelyophilization formulation for therapeutic agents. In some embodiments,the present formulations provide aqueous formulations for therapeuticagents. In some embodiments the formulations are stable formulations.

Stable Formulations

As used herein, the term “stable” refers to the ability of thetherapeutic agent (e.g., a recombinant enzyme) to maintain itstherapeutic efficacy (e.g., all or the majority of its intendedbiological activity and/or physiochemical integrity) over extendedperiods of time. The stability of a therapeutic agent, and thecapability of the pharmaceutical composition to maintain stability ofsuch therapeutic agent, may be assessed over extended periods of time(e.g., preferably for at least 1, 3, 6, 9, 12, 18, 24, 30, 36 months ormore). In the context of a formulation a stable formulation is one inwhich the therapeutic agent therein essentially retains its physicaland/or chemical integrity and biological activity upon storage andduring processes (such as freeze/thaw, mechanical mixing andlyophilization). For protein stability, it can be measure by formationof high molecular weight (HMW) aggregates, loss of enzyme activity,generation of peptide fragments and shift of charge profiles.

Stability of the therapeutic agent is of particular importance withrespect to the maintenance of the specified range of the therapeuticagent concentration required to enable the agent to serve its intendedtherapeutic function. Stability of the therapeutic agent may be furtherassessed relative to the biological activity or physiochemical integrityof the therapeutic agent over extended periods of time. For example,stability at a is time point may be compared against stability at anearlier time point (e.g., upon formulation day 0) or againstunformulated therapeutic agent and the results of this comparisonexpressed as a percentage. Preferably, the pharmaceutical compositionsof the present invention maintain at least 100%, at least 99%, at least98%, at least 97% at least 95%, at least 90%, at least 85%, at least80%, at least 75%, at least 70%, at least 65%, at least 60%, at least55% or at least 50% of the therapeutic agent's biological activity orphysiochemical integrity over an extended period of time (e.g., asmeasured over at least about 1-36 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 18, 24, 30, or 36) months, at room temperature, 2-8° C.or under accelerated storage conditions).

The therapeutic agents are preferably soluble in the pharmaceuticalcompositions of the present invention. The term “soluble” as it relatesto the therapeutic agents of the present invention refer to the abilityof such therapeutic agents to form a homogenous solution. Preferably thesolubility of the therapeutic agent in the solution into which it isadministered and by which it is transported to the target site of action(e.g., the cells and tissues of the brain) is sufficient to permit thedelivery of a therapeutically effective amount of the therapeutic agentto the targeted site of action. Several factors can impact thesolubility of the therapeutic agents. For example, relevant factorswhich may impact protein solubility include ionic strength, amino acidsequence and the presence of other co-solubilizing agents or salts(e.g., calcium salts.) In some embodiments, the pharmaceuticalcompositions are formulated such that calcium salts are excluded fromsuch compositions.

Suitable formulations, in either aqueous, pre-lyophilized, lyophilizedor reconstituted form, may contain a therapeutic agent of interest atvarious concentrations. In some embodiments, formulations may contain aprotein or therapeutic agent of interest at a concentration in the rangeof about 0.1 mg/ml to 100 mg/ml (e.g., about 0.1 mg/ml to 80 mg/ml,about 0.1 mg/ml to 60 mg/ml, about 0.1 mg/ml to 50 mg/ml, about 0.1mg/ml to 40 mg/ml, about 0.1 mg/ml to 30 mg/ml, about 0.1 mg/ml to 25mg/ml, about 0.1 mg/ml to 20 mg/ml, about 0.1 mg/ml to 60 about 0.1mg/ml to 50 mg/ml, about 0.1 mg/ml to 40 mg/ml, about 0.1 mg/ml to 30mg/ml, about 0.1 mg/ml to 25 mg/ml, about 0.1 mg/ml to 20 about 0.1mg/ml to 15 mg/ml, about 0.1 mg/ml to 10 mg/ml, about 0.1 mg/ml to 5mg/ml, about 1 mg/ml to 10 mg/ml, about 1 mg/ml to 20 mg/ml, about 1mg/ml to 40 mg/ml, about 5 mg/ml to 100 mg/ml, about 5 mg/ml to 50mg/ml, or about 5 mg/ml to 25 mg/ml). In some embodiments, formulationsaccording to the invention may contain a therapeutic agent at or above aconcentration of approximately 1 mint 5 mg/nil, 10 mg/ml, 15 mg/ml, 20mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 55mg/ml, 60 mg/ml, 65 mg/ml, 70 mg/ml, 75 mg/ml, 80 mg/ml, 85 mg/nil, 90mg/ml, 95 mg/ml, or 100 mg/ml.

The formulations of the present invention are characterized by theirtolerability either as aqueous solutions or as reconstituted lyophilizedsolutions. As used herein, the terms “tolerable” and “tolerability”refer to the ability of the pharmaceutical compositions of the presentinvention to not elicit an adverse reaction in the subject to whom suchcomposition is administered, or alternatively not to elicit a seriousadverse reaction in the subject to whom such composition isadministered. In some embodiments, the pharmaceutical compositions ofthe present invention are well tolerated by the subject to whom suchcompositions is administered.

Many therapeutic agents, and in particular the proteins and enzymes ofthe present invention, require controlled pH and specific excipients tomaintain their solubility and stability in the pharmaceuticalcompositions of the present invention. Table 4 below identifies typicalaspects of protein formulations considered to maintain the solubilityand stability of the protein therapeutic agents of the presentinvention.

TABLE 4 Parameter Typical Range/Type Rationale pH 5 to 7.5 For stabilitySometimes also for solubility Buffer type acetate, succinate, Tomaintain optimal pH citrate, histidine, May also affect stabilityphosphate or Tris Buffer 5-50 mM To maintain pH concentration May alsostabilize or add ionic strength Tonicifier NaCl, sugars, To renderiso-osmotic or isotonic mannitol solutions Surfactant Polysorbate 20, Tostabilize against interfaces and polysorbate 80 shear Other Amino acids(e.g. For enhanced solubility or stability arginine) at tens to hundredsof mM

Buffers

The pH of the formulation, is an additional factor which is capable ofaltering the solubility of a therapeutic agent e.g., an enzyme orprotein) in an aqueous formulation or for a pre-lyophilizationformulation. Accordingly, in some embodiments, formulations of thepresent invention may comprise one or more buffers. In some embodimentsthe aqueous formulations comprise an amount of buffer sufficient tomaintain the optimal pH of said composition between about 4.0-8.0 (e.g.,about 4.0, 4.5, 5.0, 5.5, 6.0, 6.2, 6.4, 6.5, 6.6, 6.8, 7.0, 7.5, or8.0). In some embodiments, the pH of the formulation is between aboutbetween about 55-7.0, between about 6.0-7.0, between about 5.5-6.0,between about 5.5-6.5, between about 5.0-6.0, between about 5.0-6.5 andbetween about 6.0-75, Suitable buffers include, for example acetate,citrate, histidine, phosphate, succinate,tris(hydroxymethyl)aminomethane (“Tris”) and our organic acids. Thebuffer concentration and pH range of the pharmaceutical compositions ofthe present invention are factors in controlling or adjusting thetolerability of the formulation. In some embodiments, a buffering agentis present at a concentration ranging between about 1 mM to about 150mM, or between about 10 mM to about 50 mM, or between about 15 mM toabout 50 mM, or between about 20 mM to about 50 mM, or between about 25mM to about 50 mM. In some embodiments, a suitable buffering agent ispresent at a concentration of approximately 1 mM, 5 mM, 10 mM, 0.15 mM,20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 75 mM, 100 mM, 125 mMor 150 mM.

In other embodiments, formulations according, to the present inventioncontain no buffers. As used herein, a formulation without buffer isreferred to as saline-based formulations.

Tonicity

In some embodiments, formulations, in either aqueous, pre-lyophilized,lyophilized or reconstituted form, contain an isotonicity agent to keepthe formulations isotonic. Typically, by “isotonic” is meant that theformulation of interest has essentially the same osmotic pressure ashuman blood. Isotonic formulations will generally have an osmoticpressure from about 240 mOsm/kg to about 350 mOsm/kg. Isotonicity can bemeasured using, for example, a vapor pressure or freezing point typeosmometers. Exemplary isotonicity agents include, but are not limitedto, glycine, sorbitol, mannitol, sodium chloride and asinine. In someembodiments, suitable isotonic agents may be present in aqueous and/orpre-lyophilized formulations at a concentration from about 0.01-5%(e.g., 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0,2.5, 3.0, 4.0 or 5.0%) by weight. In some embodiments, formulations forlyophilization contain an isotonicity agent to keep thepre-lyophilization formulations or the reconstituted formulationsisotonic.

While generally isotonic solutions are preferred for parenterallyadministered drugs, the use of isotonic solutions may change solubilityfor some therapeutic agents and in particular some proteins and/orenzymes. Slightly hypertonic solutions (e.g., up to 175 mM sodiumchloride in 5 mM sodium phosphate at pH 7.0) and sugar-containingsolutions (e.g., up to 2% sucrose in 5 mM sodium phosphate at pH 7.0)have been demonstrated to be well tolerated. The most common approvedCNS bolus formulation composition is saline (about 150 mM NaCl inwater).

Stabilizing Agents

In some embodiments, formulations may contain a stabilizing agent, orlyoprotectant, to protect the protein. Typically, a suitable stabilizingagent is a sugar, a non-reducing sugar and/or an amino acid. Exemplarysugars include, but are not limited to, dextran, lactose, mannitol,mannose, sorbitol, raffinose, sucrose and trehalose. Exemplary aminoacids include, but are not limited to, arginine, glycine and methionine.Additional stabilizing agents may include sodium chloride, hydroxyethylstarch and polyvinylpyrolidone. The amount of stabilizing agent in thelyophilized formulation is generally such that the formulation will beisotonic. However, hypertonic reconstituted formulations may also besuitable. In addition, the amount of stabilizing agent must not be toolow such that an unacceptable amount of degradation/aggregation of thetherapeutic agent occurs. Exemplary stabilizing agent concentrations inthe formulation may range from about mM to about 400 mM (e.g., fromabout 30 mM to about 300 mM, and from about 50 mM to about 100 mM), oralternatively, from 0.1% to 15% (e.g., from 1% to 10%, from 5% to 15%,from 5% to 10%) by weight. In some embodiments, the ratio of the massamount of the stabilizing agent and the therapeutic agent is about 1:1.In other embodiments, the ratio of the mass amount of the stabilizingagent and the therapeutic agent can be about 0.1:1, 0.2:1, 0.25:1,0.4:1, 0.5:1, 1:1, 2:1, 2.6:1, 3:1, 4:1, 5:1, 10:1, or 20:1. In someembodiments, suitable for lyophilization, the stabilizing agent is alsoa lyoprotectant.

In some embodiments, liquid formulations suitable for the presentinvention contain amorphous materials. In some embodiments, liquidformulations suitable for the present invention contain a substantialamount of amorphous materials (e.g., sucrose-based formulations). Insome embodiments, liquid formulations suitable for the present inventioncontain partly crystalline/partly amorphous materials.

Bulking Agents

In some embodiments, suitable formulations for lyophilization may antherinclude one or more bulking agents. A “bulking agent” is a compoundwhich adds mass to the lyophilized mixture and contributes to thephysical structure of the lyophilized cake. For example, a bulking agentmay improve the appearance of lyophilized cake (e.g., essentiallyuniform lyophilized cake). Suitable bulking agents include, but are notlimited to, sodium chloride, lactose, mannitol, glycine, sucrose,trehalose, hydroxyethyl starch. Exemplary concentrations of bulkingagents are from about 1% to about 10% (e.g., 1.0%, 1.5%, 2.0%, 2.5%,3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%,9.0%, 9.5%, and 10.0%).

Surfactants

In some embodiments, it is desirable to add a surfactant toformulations. Exemplary surfactants include nonionic surfactants such asPolysorbates (e.g., Polysorbates 20 or 80); poloxamers (e.g., poloxamer188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., Pluronics, PF68, etc).

Poloxamer

In some embodiments, formulations provided by the present inventioncontain a poloxamer as a surfactant. Typically, poloxamers arenon-ionic, triblock copolymers. Poloxamers typically have an amphiphilicstructure comprised of as central hydrophobic block, between twohydrophilic blocks. Generally, the central hydrophobic block ispolyoxypropylene (poly(propylene oxide)) (“PPO”) and the two hydrophilicblocks are polyoxyethylene (poly(ethylene oxide)) (“PEO”). A generalstructure for an exemplary poloxamer is shown as follows:

Poloxamers are also known by the trade name Pluronics® (manufactured byBASF). Poloxamers/Pluronics® are available in various grades differingin molecular weights, ratios of hydrophilic to hydrophobic blocks andphysical forms, (i.e., liquid, flakes/solids or paste). Exemplarypoloxamers/Pluronics® products suitable for the present invention areshown in Table 5 below.

TABLE 5 Poloxamer/Pluronic ® grades and their chemical compositionContent of Oxyethylene Pluronic ® Poloxamer a b (Percent) Molecularweight L 44 NF 124 12 20 44.8-48.6 2090-2360 F 68 NF 188 79 28 79.9-83.77680-9510 F 87 NF 237 64 37 70.5-74.3 6840-8830 F 108 NF 338 141 4481.4-84.9 12700-17400 F 127 NF 407 101 56 71.5-74.9  9840-14600

In some embodiments, a poloxamer suitable for the present invention isPoloxamer 188 (e.g., Plutonic® F-68). Other suitable poloxamers include,but are not limited to, Poloxamers 101, 105, 108, 122, 123, 124, 181,182, 183, 184, 185, 188, 212, 215, 217, 231, 234, 215, 237, 238, 282,284, 288, 331, 333, 334, 335, 338, 401, 402, 403, 407, and combinationthereof.

A surfactant can be included in a formulation of the invention atvarious concentrations. In particular, a surfactant may be present in aformulation at a concentration of approximately 0.05%, 0.06%, 0.07%,0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%,0.18%, 0.19%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%,0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, or 1.0%, etc. In someembodiments, a surfactant is present at a concentration rangingapproximately between 0,001% and (e.g., between 0.001% and 0.8%, between0.001% and 0.6%, between 0.001% and 0.5%, between 0.001% and 0.4%,between 0.001% and 0.3%, between 0.001% and 0.2%, between 0.005% and0.05%, between 0.005% and 0.02%, between 0.05% and 1.0%, between 0.05%and 0.75%, between 0.05% and 0.5%, between 0.05% and 0.4%, between 0.05%and 0.3%, between 0.05 and 0.2%, or between 0.05% and 0.15%) by weight.

Alternatively, or in addition, the surfactant may be added to thelyophilized formulation, pre-lyophilized formulation and/or thereconstituted formulation.

Typically, the amount of surfactant in a formulation is such that itreduces aggregation of the protein and minimizes the formation ofparticulates or effervescences. For example, a surfactant may be presentin an amount such that aggregation, particulate formation and/oreffervescences is decreased by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 803, 85%, 90%, 95%, or more as compared toa control formulation lacking the surfactant. A formulation inaccordance with the present invention may be one wherein less than about10% (e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%) andpreferably less than about (e.g., less than 4%, 3%, 2%, 1%, 0.5%) of theprotein is present as an aggregate in the formulation also referred toas high molecular weight species (“HMW”).

In some embodiments, the present invention provides “stable”formulations for lysosomal enzymes described herein. As used herein, theterm “stable” refers to the ability of the therapeutic agent (e.g., arecombinant enzyme) to maintain its therapeutic efficacy (e.g., all orthe majority of its intended biological activity and/or physiochemicalintegrity) over extended periods of time. The stability of a therapeuticagent, and the capability of the pharmaceutical composition to maintainstability of such therapeutic agent, may be assessed over extendedperiods of time (e.g., for at least 1, 3, 6, 12, 18, 24, 30, 34 monthsor more). In general, pharmaceutical compositions described herein havebeen formulated such that they are capable of stabilizing, oralternatively slowing or preventing the degradation, of one or moretherapeutic agents formulated therewith (e.g., recombinant proteins), inthe context of a formulation, a stable formulation is one in which thetherapeutic agent therein essentially retains its physical and/orchemical integrity and biological activity upon storage and duringprocesses (such as freeze/thaw, mechanical mixing and lyophilization).For protein stability, it can be measured by formulation of highmolecular weight (HMW) aggregates, loss of enzyme activity, generationof peptide fragments and shift of charge profiles. Among other things,formulations provided by the present invention reduce or eliminateformation of high molecule weight aggregates, protein degradation duringfreeze-drying, storage, shipping and infusion. In particularembodiments, a stable formulation provided by the present inventionreduces or eliminates enzyme precipitation during freeze-drying,storage, Shipping and infusion.

Stability can be measured after storage at a selected temperature (e.g.,0° C., 5° C., 25° C. (room temperature), 30° C., 40° C.) for a selectedtime period (e.g., 2 weeks, 1 month, 1.5 months, 2 months, 3 months, 4months, 5 months, 6 months, 12 months, 18 months, 24 months, etc.). Forrapid screening, the formulation may be kept at 40° C. for 2 weeks to 1month, at which time stability is measured. Where the formulation is tobe stored at 2-8° C., generally the formulation should be stable at 25°C. (i.e., room temperature) or 40° C. for at least 1 month and/or stableat 2-8° C. for at least 3 months, 6 months, 1 year or 2 years. Where theformulation is to be stored at 30° C., generally the formulation shouldbe stable for at least 3 months, 6 months, 1 year or 2 years at 30° C.and/or stable at 40° C. for at least 2 weeks, 1 month, 3 months or 6months. In some embodiments, the extent of aggregation followinglyophilization and storage can be used as an indicator of proteinstability. As used herein, the term “high molecular weight (“HMW”)aggregates” refers to an association of at least two protein monomers.For the purposes of this invention, a monomer refers to the single unitof any biologically active form of the protein of interest. Theassociation may be covalent, non-covalent, disulfide, non-reduciblecrosslinking, or by other mechanism.

In some embodiments, aggregation and/or particulate formation can bemeasured after shaking the formulation (e.g., for 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours or more). Insome embodiments, aggregation and/or particulate formation can bemeasured after pumping the formulation at high speed (e.g., through anIVEC pump). In some embodiments, aggregation and/or particulateformation can be verified visually after pumping the formulation at highspeed and shaking the formulation. In some embodiments, aggregationand/or particulate formation can be measured by an increase in aggregateformation following lyophilization and storage of the lyophilizedformulation.

Other pharmaceutically acceptable carriers, excipients or stabilizerssuch as those described in Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980) may be included in the formulation (and/orthe lyophilized formulation and/or the reconstituted formulation)provided that they do not adversely affect the desired characteristicsof the formulation. Acceptable carriers, excipients or stabilizers arenontoxic to recipients at the dosages and concentrations employed andinclude, but are not limited to, additional buffering agents;preservatives; co-solvents; antioxidants including ascorbic acid andmethionine; chelating agents such as EDTA; metal complexes (e.g.,Zn-protein complexes); biodegradable polymers such as polyesters; and/orsalt-forming counter-ions such as sodium.

Formulations, in either aqueous, pre-lyophilized, lyophilized orreconstituted form, in accordance with the present invention can beassessed based on product quality analysis, reconstitution time (iflyophilized), quality of reconstitution if lyophilized), high molecularweight, moisture, and glass transition temperature. Typically, proteinquality and product analysis, including product degradation rateanalysis and protein aggregation analysis, may use methods including,but not limited to size exclusion HPLC (SE-HPLC), cation exchange-HPLC(CEX-HPLC), X-ray diffraction (XRD), modulated differential scanningcalorimetry (mDSC), reversed phase HPLC(RP-HPLC), multi-angle lightscattering (MALS), fluorescence, ultraviolet absorption, nephelometry,capillary electrophoresis (CE), SDS-PAGE, and combinations thereof. Insome embodiments, evaluation of product in accordance with the presentinvention may include a step of evaluating appearance (either liquid orcake appearance). In some embodiments, aggregation is determined byvisual examination.

Generally, formulations (lyophilized or aqueous) can be stored forextended periods of time at room temperature. Storage temperature maytypically range from 0° C. to 45° C. (e.g., 4° C., 20° C., 25° C., 45°C. etc.). Formulations may be stored for a period of months to a periodof years. Storage time generally will be 2.4 months, 12 months, 6months, 4.5 months, 3 months, 2 months or 1 month. Formulations can bestored directly in the container used for administration, eliminatingtransfer steps.

In some embodiments, a “stable” lyophilized formulation may be onewherein the increase in aggregate in the lyophilized formulation is lessthan about 5% (e.g., less than 4%, 3%, 2%, 0.5%) and preferably lessthan about 3% (e.g., 2%, 1%, 0.5%, 0.2%, 0.1%) when the lyophilizedformulation is stored at 25° C. (i.e., room temperature) or 40° C. forat least 2 weeks, 1 month, 3 months or 6 months, or at 2-8° C. for atleast 3 months, 6 mouths, 1 year or 2 years.

Formulations can be stored directly in the lyophilization container (iflyophilized), which may also function as the reconstitution vessel,eliminating transfer steps. Alternatively, lyophilized productformulations may be measured into smaller increments for storage.Storage should generally avoid circumstances that lead to degradation ofthe proteins, including but not limited to exposure to sunlight, UVradiation, other forms of electromagnetic radiation, excessive heat orcold, rapid thermal shock, and mechanical shock.

Lyophilization

Inventive methods in accordance with the present invention can beutilized to lyophilize any materials, in particular, therapeutic agents.Typically, a pre-lyophilization formulation further contains anappropriate choice of excipients or other components such asstabilizers, buffering agents, bulking agents, and surfactants toprevent compound of interest from degradation (e.g., proteinaggregation, deamidation, and/or oxidation) during freeze-drying andstorage. The formulation for lyophilization can include one or moreadditional ingredients including lyoprotectants or stabilizing agents,buffers, bulking agents, isotonicity agents and surfactants.

After the substance of interest and any additional components are mixedtogether, the formulation is lyophilized. Lyophilization generallyincludes three main stages: freezing, primary drying and secondarydrying. Freezing is necessary to convert water to ice or some amorphousformulation components to the crystalline form. Primary drying is theprocess step when ice is removed from the frozen product by directsublimation at low pressure and temperature. Secondary drying is theprocess step when bounded water is removed from the product matrixutilizing the diffusion of residual water to the evaporation surface.Product temperature during secondary drying is normally higher thanduring primary drying. See, Tang X. et al. (2004) “Design offreeze-drying processes for pharmaceuticals: Practical advice,” Pharm.Res., 21:191-200; Nail S. L. et al. (2002) “Fundamentals offreeze-drying,” in Development and manufacture of proteinpharmaceuticals. Nail S. L. editor New York: Kluwer Academic/PlenumPublishers, pp 281-353; Wang et al. (2000) “Lyophilization anddevelopment of solid protein Pharmaceuticals,” Int. J. Pharm., 203;1-60; Williams N. A. et al. (1984) “The lyophilization ofpharmaceuticals; A literature review,” J. Parenteral Sci. Technol.,38:48-59. Generally, any lyophilization process can be used inconnection with the present invention.

In some embodiments, an annealing step may be introduced during theinitial freezing of the product. The annealing step may reduce theoverall cycle time. Without wishing to be bound by any theories, it iscontemplated that the annealing step can help promote excipientcrystallization and formation of larger ice crystals due tore-crystallization of small crystals formed during supercooling, which,in turn, improves reconstitution. Typically, an annealing step includesan interval or oscillation in the temperature during freezing. Forexample, the freeze temperature may be −40° C., and the annealing stepwill increase the temperature to, for example, −10° C. and maintain thistemperature for a set period of time. The annealing step time may rangefrom 0.5 hours to 8 hours (e.g., 0.5, 1.0 1.5, 2.0, 2.5, 3, 4, 6, and 8hours. The annealing temperature may be between the freezing temperatureand 0° C.

Lyophilization may be performed in a container, such as a tube, a bag, abottle, a tray, a vial (e.g., a glass vial), syringe or any othersuitable containers. The containers may be disposable. Lyophilizationmay also be performed in a large scale or small scale. In someinstances, it may be desirable to lyophilize the protein formulation inthe container in which reconstitution of the protein is to be carriedout in order to avoid as transfer step. The container in this instancemay, for example, be a 3, 4, 5, 10, 20, 50 or 100 cc vial.

Many different freeze-dryers are available for this purpose such as Hullpilot scale dryer (SP Industries, USA), Genesis (SP industries)laboratory freeze-dryers, or any freeze-dryers capable of controllingthe given lyophilization process parameters. Freeze-drying isaccomplished by freezing the formulation and subsequently subliming icefrom the frozen content at a temperature suitable for primary drying.Initial freezing brings the formulation to a temperature below about−20° C. (e.g., −50° C., −45° C., −40° C., −35° C., −30° C., −25° C.,etc.) in typically not more than about 4 hours (e.g., not more thanabout 3 hours, not more than about 2.5 hours, not more than about 2hours). Under this condition, the product temperature is typically belowthe eutectic point or the collapse temperature of the formulation.Typically, the shelf temperature for the primary drying will range fromabout −30 to 25° C. (provided the product remains below the meltingpoint during primary drying) at a suitable pressure, ranging typicallyfrom about 20 to 250 nTorr. The formulation, size and type of thecontainer holding the sample (e.g., glass vial) and the volume of liquidwill mainly dictate the time required for drying, which can range from afew hours to several days. A secondary drying stage is carried out atabout 0-60° C., depending primarily on the type and size of containerand the type of therapeutic protein employed. Again, volume of liquidwill mainly dictate the time required for drying, which can range from afew hours to several days.

As a general proposition, lyophilization will result in a lyophilizedformulation in which the moisture content thereof is less than about 5%,less than about 4% less than about 3%, less than about 2%, less thanabout 1%, and less than about 0.5%,

Reconstitution

While the pharmaceutical compositions of the present invention aregenerally in an aqueous form upon administration to a subject, in someembodiments the pharmaceutical compositions of the present invention arelyophilized. Such compositions must be reconstituted by adding one ormore diluents thereto prior to administration to as subject. At thedesired stage, typically at an appropriate time prior to administrationto the patient, the lyophilized formulation may be reconstituted with adiluent such that the protein concentration in the reconstitutedformulation is desirable.

Various diluents may be used in accordance with the present invention,in some embodiments, a suitable diluent for reconstitution is water. Thewater used as the diluent can be treated in a variety of ways includingreverse osmosis, distillation, deionization, filtrations (e.g.,activated carbon, microfiltration, nanofiltration) and combinations ofthese treatment methods. In general, the water should be suitable forinjection including, but not limited to, sterile water or bacteriostaticwater for injection.

Additional exemplary diluents include a pH buffered solution (e.g.,phosphate buffered saline), sterile saline solution, Elliot's solution,Ringers solution or dextrose solution. Suitable diluents may optionallycontain a preservative. Exemplary preservatives include aromaticalcohols such as benzyl or phenol alcohol. The amount of preservativeemployed is determined by assessing different preservativeconcentrations for compatibility with the protein and preservativeefficacy testing. For example, if the preservative is an aromaticalcohol (such as benzyl alcohol), it can be present in an amount fromabout 0.1-2.0%, from about 0.5-1.5%, or about 1.0-1.2%.

Diluents suitable for the invention may include a variety of additives,including, but not limited to, pH buffering agents, (e.g. Tris,histidine,) salts (e.g., sodium chloride) and other additives (e.g.,sucrose) including those described above (e.g. stabilizing agents,isotonicity agents).

According to the present invention, a lyophilized substance (e.g.,protein) can be reconstituted to a concentration of at least 25 mg/ml(e.g. at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml) and inany ranges therebetween. In some embodiments, a lyophilized substance(e.g., protein) may be reconstituted to a concentration ranging fromabout 1 mg/ml to 100 mg/ml (e.g., from about 1 mg/ml to 50 mg/ml, from 1mg/ml to 100 mg/ml, from about 1 mg/ml to about 5 mg/ml, from about 1mg/ml to about 10 mg/ml, from about 1 to about 25 mg/ml, from about 1mg/ml to about 75 mg/ml, from about 10 mg/ml to about 30 mg/ml, fromabout 10 mg/ml to about 50 mg/ml, from about 10 mg/ml to about 75 mg/ml,from about 10 mg/ml to about 100 mg/ml, from about 25 mg/nit to about 50mg/ml, from about 25 mg/ml to about 7.5 mg/ml, from about 25 mg/ml toabout 100 mg/ml, from about 50 mg/ml to about 75 mg/ml, from about 50mg/ml to about 100 mg/ml). In some embodiments, the concentration ofprotein in the reconstituted formulation may be higher than theconcentration in the pre-lyophilization formulation. High proteinconcentrations in the reconstituted formulation are considered to beparticularly useful where subcutaneous or intramuscular delivery of thereconstituted formulation is intended. In some embodiments, the proteinconcentration in the reconstituted formulation may be about 2-30 times(e.g., about 2-20, about 2-10 times, or about 2-5 times) of thepre-lyophilized formulation. In some embodiments, the proteinconcentration in the reconstituted formulation may be at least about 2times (e.g., at least about 3, 4, 5, 10, 20, 40 times) of thepre-lyophilized formulation.

Reconstitution according to the present invention may be performed inany container. Exemplary containers suitable for the invention include,but are not limited to, such as tubes, vials, syringes (e.g.,single-chamber or dual-chamber), bags, bottles, and trays. Suitablecontainers may be made of any materials such as glass, plastics, metal.The containers may be disposable or reusable. Reconstitution may also beperformed in a large scale or small scale.

In some instances, it may be desirable to lyophilize the proteinformulation in the container in which reconstitution of the protein isto be carried out in over to avoid a transfer step. The container inthis instance may, for example, be a 3, 4, 5, 10, 20, 50 or 100 cc vial.In some embodiments, a suitable container for lyophilization andreconstitution is a dual chamber syringe (e.g., Lyo-Ject,® (Vetter)syringes). For example, a dual chamber syringe may contain both thelyophilized substance and the diluent, each in a separate chamber,separated by a stopper (see Example 5). To reconstitute, a plunger canbe attached to the stopper at the diluent side and pressed to movediluent into the product chamber so that the diluent can contact thelyophilized substance and reconstitution may take place as describedherein (see Example 5).

The pharmaceutical compositions, formulations and related methods of theinvention are useful for delivering a variety of therapeutic agents tothe CNS of a subject (e.g., intrathecally, intraventricularly orintracisternally) and for the treatment of the associated diseases. Thepharmaceutical compositions of the present invention are particularlyuseful for delivering proteins and enzymes (e.g., enzyme replacementtherapy) to subjects suffering from lysosomal storage disorders. Thelysosomal storage diseases represent a group of relatively rareinherited metabolic disorders that result from defects in lysosomalfunction. The lysosomal diseases are characterized by the accumulationof undigested macromolecules within the lysosomes, which results in anincrease in the size and number of such lysosomes and ultimately incellular dysfunction and clinical abnormalities.

CNS Delivery

It is contemplated that various stable formulations described herein aregenerally suitable for CNS delivery of therapeutic agents. Stableformulations according to the present invention can be used for CNSdelivery via various techniques and routes including, but not limitedto, intraparenchymal, intracerebral, intravetricular cerebral (ICV),intrathecal (e.g., IT-Lumbar, IT-cisterna magna) administrations and anyother techniques and routes for injection directly or indirectly to theCNS and/or CSF. Exemplary CNS delivery of stable formulations aredescribed in PCT/US2011/041926, filed on Jun. 25, 2011 entitled “Methodsand Compositions for CNS Delivery of Arylsulfatase A,” the entirecontents of Which are incorporated herein by reference.

Intrathecal Delivery

In some embodiments, a replacement enzyme is delivered to the CNS in aformulation described herein. In some embodiments, a replacement enzymeis delivered to the CNS by administering into the cerebrospinal fluid(CSF) of a subject in need of treatment. In some embodiments,intrathecal administration is used to deliver a desired replacementenzyme (e.g., an ASA protein) into the CSF. As used herein, intrathecaladministration (also referred to as intrathecal injection) refers to aninjection into the spinal canal (intrathecal space surrounding thespinal cord). Various techniques may be used including, withoutlimitation, lateral cerebroventricular injection through a burrhole orcistemal or lumbar puncture or the like. Exemplary methods are describedin Lazorthes et al. Advances in Drug Delivery Systems and Applicationsin Neurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1:169-179, the contents of which are incorporated herein by reference.

According to the present invention, an enzyme may be injected at anyregion surrounding the spinal canal. In some embodiments, an enzyme isinjected into the lumbar area or the cisterna magna orintraventricularly into a cerebral ventricle space. As used herein, theterm “lumbar region” or “lumbar area” refers to the area between thethird and fourth lumbar (lower back) vertebrae and, more inclusively,the L2-S1 region of the spine. Typically, intrathecal injection via thelumbar region or lumber area is also referred to as “lumbar IT delivery”or “lumbar IT administration.” the term “cisterna magna” refers to thespace around and below the cerebellum via the opening between the skulland the top of the spine. Typically, intrathecal injection via cisternamagna is also referred to as “cisterna magna delivery.” The term“cerebral ventricle” refers to the cavities in the brain that arecontinuous with the central canal of the spinal cord. Typically,injections via the cerebral ventricle cavities are referred to asintravetricular Cerebral (ICV) delivery.

In some embodiments, “intrathecal administration” or “intrathecaldelivery” according to the present invention refers to lumbar ITadministration or delivery, for example, delivered between the third andfourth lumbar (lower back) vertebrae and, more inclusively, the L2-S1region of the spine. It is contemplated that lumbar IT administration ordelivery distinguishes over cisterna magna delivery in that lumbar ITadministration or delivery according to our invention provides betterand more effective delivery to the distal spinal canal, while cisternamagna delivery, among other things, typically does not deliver well tothe distal spinal canal.

Device for Intrathecal Delivery

Various devices may be used for intrathecal delivery according to thepresent invention. In some embodiments, a device for intrathecaladministration contains a fluid access port (e.g., injectable port); ahollow body (e.g., catheter) having a first flow orifice in fluidcommunication with the fluid access port and a second flow orificeconfigured for insertion into spinal cord; and a securing mechanism forsecuring the insertion of the hollow body in the spinal cord. As anon-limiting example shown in FIG. 62, a suitable securing mechanismcontains one or more knobs mounted on the surface of the hollow body anda sutured ring adjustable over the one or more knobs to present thehollow body (e.g., catheter) from slipping out of the spinal cord. Invarious embodiments, the fluid access port comprises a reservoir. Insome embodiments, the fluid access port comprises a mechanical pump(e.g., an infusion pump). In some embodiments, an implanted catheter isconnected to either a reservoir (e.g., for bolus delivery), or aninfusion pump. The fluid access port may be implanted or external

In some embodiments, intrathecal administration may be performed byeither lumbar puncture (i.e., slow bolus) or via a port-catheterdelivery system (i.e., infusion or bolus). In some embodiments, thecatheter is inserted between the laminae of the lumbar vertebrae and thetip is threaded up the thecal space to the desired level (generallyL3-L4) (FIG. 63).

Relative to intravenous administration, a single dose volume suitablefor intrathecal administration is typically small. Typically,intrathecal delivery according to the present invention maintains thebalance of the composition of the CST as well as the intracranialpressure of the subject. In some embodiments, intrathecal delivery isperformed absent the corresponding removal of CSF from a subject. Insome embodiments, a suitable single dose volume may be e.g., less thanabout 10 ml, 8 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1.5 ml, 1 ml, or 0.5nil. In some embodiments, a suitable single dose volume may be about0.5-5 ml, 0.5-4 ml, 0.5-3 ml, 0.5-2 ml, 0.5-1 ml, 1-3 ml, 1-5 ml, 1.5-3ml, 1-4 ml, or 0.5-1.5 ml. In some embodiments, intrathecal deliveryaccording to the present invention involves a step of removing a desiredamount of CSF first. In some embodiments, less than about 10 ml (e.g.,less than about 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml) ofCSF is first removed before IT administration. In those cases, asuitable single dose volume may be e.g., more than about 3 ml, 4 ml, 5ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml.

Various other devices may be used to effect intrathecal administrationof a therapeutic composition. For example, formulations containingdesired enzymes may be given using an Ommaya reservoir which is incommon use for intrathecally administering drugs for meningealcarcinomatosis (Lancet 2: 983-84, 1963). More specifically, in thismethod, a ventricular tube is inserted through a hole formed in theanterior horn and is connected to an Ommaya reservoir installed underthe scalp, and the reservoir is subcutaneously punctured tointrathecally deliver the particular enzyme being replaced, which isinjected into the reservoir. Other devices for intrathecaladministration of therapeutic compositions or formulations to anindividual are described in U.S. Pat. No. 6,217,552, incorporated hereinby reference. Alternatively, the drug may be intrathecally given, forexample, by a single injection, or continuous infusion. It should beunderstood that the dosage treatment may be in the form of a single doseadministration or multiple doses.

For injection, formulations of the invention can be formulated in liquidsolutions. In addition, the enzyme may be formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms arealso included. The injection can be, for example, in the form of a bolusinjection or continuous infusion (e.g., using infusion pumps) of theenzyme.

In one embodiment of the invention, the enzyme is administered bylateral cerebro ventricular injection into the brain of a subject. Theinjection can be made, for example, through a burr hole made in thesubject's skull. In another embodiment, the enzyme and/or otherpharmaceutical formulation is administered through a surgically insertedshunt into the cerebral ventricle of a subject. For example, theinjection can be made into the lateral ventricles, which are larger. Insome embodiments, injection into the third and fourth smaller ventriclescan also be made.

In yet another embodiment, the pharmaceutical compositions used in thepresent invention are administered by injection into the cisterna magna,or lumbar area of a subject.

In another embodiment of the method of the invention, thepharmaceutically acceptable formulation provides sustained delivery,e.g., “slow release” of the enzyme or other pharmaceutical compositionused in the present invention, to a subject for at least one, two,three, four weeks or longer periods of time after the pharmaceuticallyacceptable formulation is administered to the subject.

As used herein, the term “sustained delivery” refers to continualdelivery of a pharmaceutical formulation of the invention in vivo over aperiod of time following administration, preferably at least severaldays, a week or several weeks. Sustained delivery of the composition canbe demonstrated by, for example, the continued therapeutic effect of theenzyme over time (e.g., sustained delivery of the enzyme can bedemonstrated by continued reduced amount of storage granules in thesubject). Alternatively, sustained delivery of the enzyme may bedemonstrated by detecting the presence of the enzyme in vivo over time.

Delivery to Target Tissues

As discussed above, one of the surprising and important features or thepresent invention is that therapeutic agents, in particular, replacementenzymes administered using inventive methods and compositions of thepresent invention are able to effectively and extensively diffuse acrossthe brain surface and penetrate various layers or regions of the brain,including deep brain regions. In addition, inventive methods andcompositions of the present invention effectively deliver therapeuticagents (e.g., an ASA enzyme) to various tissues, neurons or cells ofspinal cord, including the lumbar region, which is hard to target byexisting CNS delivery methods such as ICV injection. Furthermore,inventive methods and compositions of the present invention deliversufficient amount of therapeutic agents (e.g., an ASA enzyme) to bloodstream and various peripheral organs and tissues.

Thus, in some embodiments, a therapeutic protein (e.g. an ASA enzyme) isdelivered to the central nervous system of a subject. In someembodiments, a therapeutic protein (e.g., an ASA enzyme) is delivered toone or more of target tissues of brain, spinal cord, and/or peripheralorgans. As used herein, the term “target tissues” refers to any tissuethat is affected by the lysosomal storage disease to be treated or anytissue in which the deficient lysosomal enzyme is normally expressed. Insome embodiments, target tissues include those tissues in which there isa detectable or abnormally high amount of enzyme substrate, for examplestored in the cellular lysosomes of the tissue, in patients suffering,from or susceptible to the lysosomal storage disease. In someembodiments, target tissues include those tissues that displaydisease-associated pathology, symptom, or feature. In some embodiments,target tissues include those tissues in which the deficient lysosomalenzyme is normally expressed at an elevated level. As used herein, atarget tissue may be a brain target tissue, a spinal cord target tissueand/or a peripheral target tissue. Exemplary target tissues aredescribed in detail below.

Brain Target Tissues

In general, the brain can be divided into different regions, layers andtissues. For example, meningeal tissue is a system of membranes whichenvelops the central nervous system, including the brain. The meningescontain three layers, including dura matter, arachnoid matter, and piamatter. In general, the primary function of the meninges and of thecerebrospinal fluid is to protect the central nervous system. In someembodiments, a therapeutic protein in accordance with the presentinvention is delivered to one or more layers of the meninges.

The brain has three primary subdivisions, including the cerebrum,cerebellum, and brain stem. The cerebral hemispheres, which are situatedabove most other brain structures and are covered with a conical layer.Underneath the cerebrum lies the brainstem, which resembles a stalk onwhich the cerebrum is attached. At the rear of the brain, beneath thecerebrum and behind the brainstem, is the cerebellum.

The diencephalon, which is located near the midline of the brain andabove the mesencephalon, contains the thalamus, metathalamus,hypothalamus, epithalamus, prethalamus, and pretectum. Themesencephalon, also called the midbrain, contains the tectum,tegumentum, ventricular mesocoelia, and cerebral peduncels, the rednucleus, and the cranial nerve III nucleus. The mesencephalon isassociated with vision, hearing, motor control, sleep/wake, alertness,and temperature regulation.

Regions of tissues of the central nervous system, including the brain,can be characterized based on the depth of the tissues. For example CNS(e.g., brain) tissues can be characterized as surface or shallowtissues, mid-depth tissues, and/or deep tissues.

According to the present invention, a therapeutic protein (e.g., areplacement enzyme) may be delivered to any appropriate brain targettissue(s) associated with a particular disease to be treated in asubject. In some embodiments, a therapeutic protein (e.g., a replacementenzyme) in accordance with the present invention is delivered to surfaceor shallow brain target tissue. In some embodiments, a therapeuticprotein in accordance with the present invention is delivered, tomid-depth brain target tissue. In some embodiments, a therapeuticprotein in accordance with the present invention is delivered to deepbrain target tissue. In some embodiments, a therapeutic protein inaccordance with the present invention is delivered to a combination ofsurface or shallow brain target tissue, mid-depth brain target tissue,and/or deep brain target tissue. In some embodiments, a therapeuticprotein in accordance with the present invention is delivered to a deepbrain tissue at least 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or morebelow (or internal to) the external surface of the brain.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more surface or shallow tissues of cerebrum. In some embodiments,the targeted surface or shallow tissues of the cerebrum are locatedwithin 4 mm from the surface of the cerebrum. In some embodiments, thetargeted surface or shallow tissues of the cerebrum are selected frompia mater tissues, cerebral cortical ribbon tissues, hippocampus,Virchow Robin space, blood vessels within the VR space, the hippocampus,portions of the hypothalamus on the inferior surface of the brain, theoptic nerves and tracts, the olfactory bulb and projections, andcombinations thereof.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more deep tissues of the cerebrum. In some embodiments, thetargeted surface or shallow tissues of the cerebrum are located 4 mm(e.g., 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm) below (or internal to)the surface of the cerebrum. In some embodiments, targeted deep tissuesof the cerebrum include the cerebral conical ribbon. In someembodiments, targeted deep tissues of the cerebrum include one or moreof the diencephalon (e.g., the hypothalamus, thalamus, prethalamus,subthalamus, etc.), metencephalon lentiform nuclei, the basal ganglia,caudate, putamen, amygdala, globus pallidus, and combinations thereof.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more tissues of the cerebellum. In certain embodiments, thetargeted one or more tissues of the cerebellum are selected from thegroup consisting of tissues of the molecular layer, tissues of thePurkinje cell layer, tissues of the Granular cell layer, cerebellarpeduncles, and combination thereof. In some embodiments, therapeuticagents (e.g., enzymes) are delivered to one or more deep tissues of thecerebellum including, but not limited to, tissues of the Purkinje celllayer, tissues of the Granular cell layer, deep cerebellar white mattertissue (e.g., deep relative to the Granular cell layer), and deepcerebellar nuclei tissue.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or mote tissues of the brainstem. In some embodiments, the targetedone or more tissues of the brainstem include brain stein white mattertissue and/or brain stem nuclei tissue.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered tovarious brain tissues including, but not limited to matter, whitematter, periventricular areas, pia-arachnoid, meninges, neocortex,cerebellum, deep tissues in cerebral cortex, molecular layer,caudate/putamen region, midbrain, deep regions of the pons or medulla,and combinations thereof.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered tovarious cells in the brain including, but not limited to, neurons, glialcells, perivascular cells and/or meningeal cells. In some embodiments, atherapeutic protein is delivered to oligodendrocytes of deep whitematter.

Spinal Cord

In general, regions or tissues of the spinal cord can be characterizedbased on the depth of the tissues. For example, spinal cord tissues canbe characterized as surface or shallow tissues, mid-depth tissues,and/or deep tissues.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more surface or shallow tissues of the spinal cord. In someembodiments, a targeted surface or shallow tissue of the spinal cord islocated within 4 mm from the surface of the spinal cord. In someembodiments, a targeted surface or shallow tissue of the spinal cordcontains pia matter and/or the tracts of white matter.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more deep tissues of the spinal cord. In some embodiments, atargeted deep tissue of the spinal cord is located internal to 4 mm fromthe surface of the spinal cord, in some embodiments, a targeted deeptissue of the spinal cord contains spinal cord grey matter and/orependymal cells.

In some embodiments, therapeutic, agents (e.g., enzymes) are deliveredto neurons of the spinal cord.

Peripheral Target Tissues

As used herein, peripheral organs or tissues refer to any organs ortissues that are not part of the central nervous system (CNS).Peripheral target tissues may include, but are not limited to, bloodsystem, liver, kidney, heart, endothelium, bone marrow and bone marrowderived cells, spleen, lung, lymph node, bone, cartilage, ovary andtestis. In some embodiments, a therapeutic protein (e.g., a replacementenzyme) in accordance with the present invention is delivered to one ormore of the peripheral target tissues.

Biodistribution and Bioavailability

In various embodiments, once delivered to the target tissue, atherapeutic agent (e.g., an ASA enzyme) is localized intracellularly.For example, a therapeutic agent (e.g., enzyme) may be localized toexons, axons, lysosomes, mitochondria or vacuoles of as target cell(e.g., neurons such as Purkinje cells). For example, in some embodimentsintrathecally-administered enzymes demonstrate translocation dynamicssuch that the enzyme moves within the perivascular space (e.g., bypulsation-assisted convective mechanisms). In addition, active axonaltransport mechanisms relating to the association of the administeredprotein or enzyme with neurofilaments may also contribute to orotherwise facilitate the distribution of intrathecally-administeredproteins or enzymes into the deeper tissues of the central nervoussystem.

In some embodiments, a therapeutic agent (e.g., an ASA enzyme) deliveredaccording to the present invention may achieve therapeutically orclinically effective levels or activities in various targets tissuesdescribed herein. As used herein, a therapeutically or clinicallyeffective level or activity is a level or activity sufficient to confera therapeutic effect in a target tissue. The therapeutic effect may beobjective (i.e., measurable by some test or marker) or subjective (i.e.,subject gives an indication of or feels an effect). For example, atherapeutically or clinically effective level or activity may be anenzymatic level or activity that is sufficient to ameliorate symptomsassociated with the disease in the target tissue (e.g., GAG storage).

In some embodiments, a therapeutic agent (e.g., as replacement enzyme)delivered according to the present invention may achieve an enzymaticlevel or activity that is at feast 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% of the normal level or activity of the correspondinglysosomal enzyme in the target tissue. In some embodiments, atherapeutic agent (e.g., a replacement enzyme) delivered according tothe present invention may achieve an enzymatic level or activity that isincreased by at least 1-fold, 2-fold, 3-fold, 4-fold, 6-fold, 7-fold,8-fold, 9-fold or 10-fold as compared to a control (e.g., endogenouslevels or activities without the treatment). In some embodiments, atherapeutic agent (e.g., a replacement enzyme) delivered according tothe present invention may achieve an increased enzymatic level oractivity at least approximately 10 nmol/hr/mg, 20 nmol/hr/mg, 40nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg, 80 nmol/hr/mg,90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200 nmol/hr/mg, 250nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 4110 nmol/hr/mg, 450nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg or 600 nmol/hr/mg in a targettissue.

In some embodiments, inventive methods according to the presentinvention are particularly useful for targeting the lumbar region. Insome embodiments, a therapeutic agent (e.g., a replacement enzyme)delivered according to the present invention may achieve an increasedenzymatic level or activity in the lumbar region of at leastapproximately 500 nmol/hr/mg, 600 nmol/hr/mg, 700 nmol/hr/mg, 800nmol/hr/mg, 900 nmol/hr/mg, 1000 nmol/hr/mg, 1500 nmol/hr/mg, 2000nmol/hr/mg, 3000 nmol/hr/mg, 4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000nmol/hr/mg, 7000 nmol/hr/mg, 8000 nmol/hr/mg, 9000 nmol/hr/mg, or 10,000nmol/hr/mg.

In general, therapeutic agents (e.g., replacement enzymes) deliveredaccording to the present invention have sufficiently long half time inCSF and target tissues of the brain, spinal cord, and peripheral organs.In some embodiments, a therapeutic agent (e.g., a replacement enzyme)delivered according to the present invention may have a half-life of atleast approximately 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 bouts, 9 hours, 10hours, 12 hours, 16 hours, 18 hours, 20 hours, 25 hours, 30 hours, 35hours, 40 hours, up to 3 days, up to 7 days, up to 14 days, up to 21days or up to a month. In some embodiments. In some embodiments, atherapeutic agent (e.g., a replacement enzyme) delivered according tothe present invention may retain detectable level or activity in CSF orbloodstream after 12 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90hours, 96 hours, 0.102 hours, or a week following administration.Detectable level or activity may be determined using, various methodsknown in the art.

In certain embodiments, a therapeutic agent (e.g., a replacement enzyme)delivered according to the present invention achieves a concentration ofat least 30 μg/ml in the CNS tissues and cells of the subject followingadministration (e.g., one week, 3 days, 48 hours, 36 hours, 24 hours, 18hours, 2 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30minutes, or less, following intrathecal administration of thepharmaceutical composition to the subject). In certain embodiments, atherapeutic agent (e.g., a replacement enzyme) delivered according tothe present invention achieves a concentration of at least 20 μg/ml, atleast 15 μg/ml, at least 10 μg/ml, at least 7.5 μg/ml, at least 5 μg/ml,at least 2.5 μg/ml, at least 1.0 μg/ml or at least 0.5 μg/ml in thetargeted tissues or cells of the subject (e.g., brain tissues orneurons) following administration to such subject (e.g., One week, 3days, 4 hours, 36 hours, 24 hours, 18 hours, 12 hours, 8 hours, 6 hours,4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or less followingintrathecal administration of such pharmaceutical compositions to thesubject).

Treatment of Metachromatic Leukodystrophy Disease (MLD)

Metachromatic Leukodystrophy Disease (MID), is an autosomal recessivedisorder resulting from a deficiency of the enzyme Arylsulfatease A(ASA). ASA, which is encoded by the ARSA gene in humans, is an enzymethat breaks down cerebroside 3-sulfate or sphingolipid3-O-sulfogalactosylceramide (sulfatide) into cerebroside and sulfate. Inthe absence of the enzyme, sulfatides accumulate in the nervous system(e.g., myelin sheaths, neurons and glial cells) and to a lesser extentin visceral organs. The consequence of these molecular and cellularevents is progressive demyelination and axonal loss within the CNS andPNS, which is accompanied clinically by severe motor and cognitivedysfunction.

A defining clinical feature of this disorder is central nervous system(CNS) degeneration, which results in cognitive impairment (e.g., mentalretardation, nervous disorders, and blindness, among others).

MLD can manifest itself in young children (Late-infantile form), whereaffected children typically begin showing symptoms just after the firstyear of life (e.g., at about 15-24 months), and generally do not survivepast the age of 5 years. MLD can manifest itself in children (Juvenileform), where affected children typically show cognitive impairment byabout the age of 3-10 years, and life-span can vary (e.g., in the rangeof 10-15 years after onset of symptoms). MLD can manifest itself inadults (Adult-onset form) and can appear in individuals of any age(e.g., typically at age 16 and later) and the progression of the diseasecan vary greatly.

Compositions and methods of the present invention may be used toeffectively treat individuals suffering from or susceptible to MLD. Theterms, “treat” or “treatment,” as used herein, refers to amelioration ofone or more symptoms associated with the disease, prevention or delay ofthe onset of one or more symptoms of the disease, and/or lessening ofthe severity or frequency of one or more symptoms of the disease.Exemplary symptoms include, but are not limited to, intracranialpressure, hydrocephalus ex vacuo, accumulated sulfated glycolipids inthe myelin sheaths in the central and peripheral nervous system and invisceral organs, progressive demyelination and axonal loss within theCNS and PNS, and/or motor and cognitive dysfunction.

In some embodiments, treatment refers to partially or completealleviation, amelioration, relief, inhibition, delaying onset, reducingseverity and/or incidence of neurological impairment in an MLD patient.As used herein, the term “neurological impairment” includes varioussymptoms associated with impairment of the central nervous system (e.g.,the brain and spinal cord). In some embodiments, various symptoms of MLDare associated with impairment of the peripheral nervous system (PNS).In some embodiments, neurological impairment in an MLD patient ischaracterized by decline in gross motor function. It will be appreciatedthat gross motor function may be assessed by any appropriate method. Forexample. In some embodiments, MISS motor function is measured as thechange from a baseline in motor function using the Gross Motor FunctionMeasure-88 (GMFM-88) total raw score.

In some embodiments, treatment refers to decreased sulfatideaccumulation in various tissues. In some embodiments, treatment refersto decreased sulfatide accumulation in brain target tissues, spinal cordneurons, and/or peripheral target tissues. In certain embodiments,sulfatide accumulation is decreased by about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100% or more as compared to a control. In some embodiments, sulfatideaccumulation is decreased by at least 1-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold as compared to acontrol. It will be appreciated that sulfatide storage may be assessedby any appropriate method. For example, in some embodiments, sulfatidestorage is measured by alcian blue staining. In some embodiments,sulfatide storage is measured by LAMP-1 staining.

In some embodiments, treatment refers to reduced vacuolization inneurons (e.g., neurons containing Purkinje cells). In certainembodiments, vacuolization in neurons is decreased by about 5%, 10%,15%, 10%, 15%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100% or more as compared to a control. In someembodiments, vacuolization is decreased by at least 1-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold as compared toa control.

In some embodiments, treatment refers to increased ASA enzyme activityin various tissues. In some embodiments, treatment refers to increasedASA enzyme activity in brain target tissues, spinal cord neurons and/orperipheral target tissues. In some embodiments, ASA enzyme activity isincreased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 49%, 45%, 50% 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%,600%, 700%, 800%, 900% 1000% or more as compared to a control. In someembodiments, ASA enzyme activity is increased by at least 1-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or10-fold as compared to a control. In some embodiments, increased ASAenzymatic activity is at least approximately 10 nmol/hr/mg, 20nmol/hr/mg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg,80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 400nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg, 600nmol/hr/mg or more. In some embodiments, ASA enzymatic activity isincreased in the lumbar region. In some embodiments, increased ASAenzymatic activity in the lumbar region is at least approximately 2000nmol/hr/mg, 3000 nmol/hr/mg, 4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000nmol/hr/mg, 7000 nmol/hr/mg, 8000 nmol/hr/mg, 9000 nmol/hr/mg, 10,000nmol/hr/mg, or more.

In some embodiments, treatment refers to decreased progression of lossof cognitive ability. In certain embodiments, progression of loss ofcognitive ability is decreased by about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% ormore as compared to a control. In some embodiments, treatment refers todecreased developmental delay. In certain embodiments, developmentaldelay is decreased by about 5%, 19%, 15%, 20%, 25%, 30%, 35%, 40%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared toa control.

In some embodiments, treatment refers to increased survival (e.g.,survival time). For example, treatment can result in an increased lifeexpectancy of a patient. In some embodiments, treatment according to thepresent invention results in an increased life expectancy of a patientby more than about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, about 100%, about 105%, about 110%, about 115%, about 120%, about125%, about 130%, about 135%, about 140%, about 145%, about 150%, about155%, about 160%, about 165%, about 170%, about 0.175%, about 0.180%,about 185%, about 190%, about 195%, about 200% or more, as compared tothe average life expectancy of one or more control individuals withsimilar disease without treatment. In some embodiments, treatmentaccording to the present invention results in an increased lifeexpectancy of a patient by more than about 6 month, about 7 months,about 8 months, about 9 months, about 10 months, about 11 months, about12 months, about 2 years, about 3 years, about 4 years, about 5 years,about 6 years, about 7 years, about 8 years, about 9 years, about 10years or more, as compared to the average life expectancy of one or morecontrol individuals with similar disease without treatment. In someembodiments, treatment according to the present invention results inlong term sum-heal of a patient. As used herein, the term “long termsurvival” refers to a survival time or life expectancy longer than about40 years, 45 years, 50 years, 55 years, 60 years or longer.

The terms, “improve,” “increase” or “reduce,” as used herein, indicatevalues that are relative to a control. In some embodiments, a suitablecontrol is a baseline measurement, such as a measurement in the sameindividual prior to initiation of the treatment described herein, or ameasurement in a control individual (or multiple control individuals) inthe absence of the treatment described herein. A “control individual” isan individual afflicted with the same form MLD (e.g., late-infantile,juvenile, or adult-onset form), who is about the same age and/or genderas the individual being treated (to ensure that the stages of thedisease in the treated individual and the control individual(s) arecomparable).

The individual (also referred to as “patient” or “subject”) beingtreated is an individual (fetus, infant, child, adolescent, or adulthuman) having, MLD or having the potential to develop WILD. Theindividual can have residual endogenous ASA expression and/or activity,or no measurable activity. For example, the individual having MLD mayhave ASA expression levels that are less than about 30-50%, less thanabout 25-30%, less than about 20-25%, less than about 15-20%, less thanabout 10-15%, less than about 5-10%, less than about 0.1-5% of normalASA expression levels.

In some embodiments, the individual is an individual who has beenrecently diagnosed with the disease. Typically, early treatment(treatment commencing as soon as possible after diagnosis) is importantto minimize the effects of the disease and to maximize the benefits oftreatment.

Immune Tolerance

Generally, intrathecal administration of a therapeutic agent (e.g.,replacement enzyme) according to the present invention does not resultin severe adverse effects in the subject. As used herein, severe adverseeffects induce, but are not limited to, substantial immune response,toxicity, or death. As used herein, the term “substantial immuneresponse” refers to severe or serious immune responses, such as adaptiveT-cell immune responses.

Thus, in many embodiments, inventive methods according to the presentinvention do not involve concurrent immunosuppressant therapy (i.e., anyimmunosuppressant therapy used as pre-treatment/pre-conditioning or inparallel to the method). In some embodiments, inventive methodsaccording to the present invention do not involve an immune toleranceinduction in the subject being treated. In some embodiments, inventivemethods according to the present invention do not involve apre-treatment or preconditioning of the subject using T-cellimmunosuppressive agent.

In some embodiments, intrathecal administration of therapeutic agentscan mount an immune response against these agents. Thus, in someembodiments, it may be useful to render the subject receiving thereplacement enzyme tolerant to the enzyme replacement therapy. Immunetolerance may be induced using various methods known in the art. Forexample, an initial 30-60 day regimen of a T-cell immunosuppressiveagent such as cyclosporin A (CsA) and an antiproliferative agent, suchas, azathioprine (Aza), combined with weekly intrathecal infusions oflow doses of a desired replacement enzyme may be used.

Any immunosuppressant agent known to the skilled artisan may be employedtogether with a combination therapy of the invention. Suchimmunosuppressant agents include but are not limited to cyclosporine,FK506, rapamycin, CTLA4-Ig, and anti-TNF agents such as etanercept (seee.g. Moder, 2000, Ann. Allergy Asthma Immunol. 84, 280-284; Nevins,2000, Curr. Opin. Pediatr. 12, 146-150; Kurlberg et al., 2000, Scand. J.Immunol. 51, 224-230; Ideguchi et al., 2000, Neuroscience 95, 217-226;Potter et al., 1999, Ann. N.Y. Acad. Sci. 875, 159-174; Slavik et al.,1999, Immunol, Res. 19, 1-24; Gaziev et al., 1999, Bone MarrowTransplant. 25, 689-696; Henry, 1999, Clin. Transplant. 13, 209-2.20;Gummert et al 1999, J. Am. Soc. Nephrol. 10, 1366-1380; Qi et al., 2000,Transplantation 69, 1275-1283). The anti-IL2 receptor (.alpha.-subunit)antibody daclizumab (e.g. Zenapax™), which has been demonstratedeffective in transplant patients, can also be used as animmunosuppressant agent (see e.g. Wiseman et al., 1999, Drugs 58,1029-1042; Beniaminovitz et al., 2000, N. Engl J. Med. 342, 613-619;Ponticelli et al., 1999, Drugs R. D. 1, 55-60; Berard et al, 1999,Pharmacotherapy 19, 1127-1137; Eckhoff et al., 2000. Transplantation 69,1867-1872; Ekberg et al., 2000, Transpl. Int. 13, 151-159), Additionalimmunosuppressant agents include but are not limited to anti-CD2 (Brancoet al., 1999, Transplantation 68, 1588-1596; Przepiorka et al., 1998,Blood 92, 4066-4071), anti-CD4 (Marinova-Mutafchieva et al., 2000,Arthritis Rheum. 43, 638-644; Fishwild et al., 1999, Clin. Immunol. 92,138-152), and anti-CD40 ligand (Hong et al., 2000, Semin, Nephrol. 20,108-125; Chirmule et. al., 2000, J. Virol. 74, 3345-3352; Ito et al.,2000, J. Immunol. 164, 1230-1235).

Administration

Inventive methods of the present invention contemplate single as well asmultiple administrations of a therapeutically effective amount of thetherapeutic agents (e.g., replacement enzymes) described herein.Therapeutic agents (e.g., replacement enzymes) can be administered atregular intervals, depending on the nature, severity and extent of thesubject's condition (e.g., a lysosomal storage disease). In someembodiments, a therapeutically effective amount of the therapeuticagents (e.g., replacement enzymes) of the present invention may beadministered intrathecally periodically at regular intervals (e.g., onceevery year, once every six months, once every five months, once everythree months, bimonthly (once every two months), monthly (once everymonth), biweekly (once every two weeks), weekly).

In some embodiments, intrathecal administration may be used inconjunction with other routes of administration (e.g., intravenous,subcutaneously, intramuscularly, parenterally, transdermally ortransmucosally (e.g., orally or nasally)). In some embodiments, thoseother routes of administration (e.g., intravenous administration) may beperformed no more frequent than biweekly, monthly, once every twomonths, once every three months, once every four months, once every fivemonths, once every six months, annually administration.

As used herein, the term “therapeutically effective amount” is largelydetermined base on the total amount of the therapeutic agent containedin the pharmaceutical compositions of the present invention. Generally,a therapeutically effective amount is sufficient to achieve a meaningfulbenefit to the subject (e.g., treating, modulating, curing, preventingand/or ameliorating the underlying disease or condition). For example, atherapeutically effective amount may be an amount sufficient to achievea desired therapeutic and/or prophylactic effect, such as an amountsufficient to modulate lysosomal enzyme receptors or their activity tothereby treat such lysosomal storage disease or the symptoms thereof(e.g., a reduction in or elimination of the presence or incidence of“zebra bodies” or cellular vacuolization following the administration ofthe compositions of the present invention to a subject). Generally, theamount of as therapeutic agent (e.g., a recombinant lysosomal enzyme)administered to a subject in need thereof will depend upon thecharacteristics of the subject. Such characteristics include thecondition, disease severity, general health, age, sex and body weight ofthe subject. One of ordinary skill in the art will be readily able todetermine appropriate dosages depending on these and other relatedfactors. In addition, both objective and subjective assays mayoptionally be employed to identify optimal dosage ranges.

A therapeutically effective amount is commonly administered in a dosingregimen that may comprise multiple unit doses. For any particulartherapeutic protein, a therapeutically effective amount (and/or anappropriate unit dose within an effective dosing regimen) may vary, forexample, depending on route of administration, on combination with otherpharmaceutical agents. Also, the specific therapeutically effectiveamount (and/or unit dose) for any particular patient may depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific pharmaceutical agentemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and/or rate of excretion or metabolism of thespecific fusion protein employed; the duration oldie treatment; and likefactors as is well known in the medical arts.

In some embodiments, the therapeutically effective dose ranges fromabout 0.005 mg/kg brain weight to 500 mg/kg brain weight, e.g., fromabout 0.005 ing/kg brain weight to 400 mg/kg brain weight, from about0.005 mg/kg brain weight to 300 mg/kg brain weight, from about 0.005mg/kg brain weight to 200 mg/kg brain weight, from about 0.005 mg/kgbrain weight to 100 mg/kg brain weight, from about 0.005 mg/kg brainweight to 90 mg/kg brain weight, from about 0.005 mg/kg brain weight to80 mg/kg brain weight, from about 0,005 mg/kg brain weight to 70 mg/kgbrain weight, from about 0.005 mg/kg brain weight to 60 mg/kg brainweight, from about 0.005 mg/kg brain weight to 50 mg/kg brain weight,from about 0.005 mg/kg brain weight to 40 mg/kg brain weight, from about0.005 mg/kg brain weight to 30 mg/kg brain weight, from about 0.005mg/kg brain weight to 25 mg/kg brain weight, from about 0.005 mg/kgbrain weight to 20 mg/kg brain weight, from about 0.005 mg/kg brainweight to 15 mg/kg brain weight, from about 0.005 mg/kg brain weight to10 mg/kg brain weight.

In some embodiments, the therapeutically effective dose is greater thanabout 0.1 mg/kg brain weight, greater than about 0.5 mg/kg brain weight,greater than about 1.0 mg/kg brain weight, greater than about 3 mg/kgbrain weight, greater than about 5 mg/kg brain weight, greater thanabout 10 mg/kg brain weight, greater than about 15 mg/kg brain weight,greater than about 20 mg/kg brain weight, greater than about 30 mg/kgbrain weight, greater than about 40 mg/kg brain weight, greater thanabout 50 mg/kg brain weight, greater than about 60 mg/kg brain weight,greater than about 70 mg/kg brain weight, greater than about 80 mg/kgbrain weight, greater than about 90 mg/kg brain weight, greater thanabout 100 mg/kg brain weight, greater than about 150 mg/kg brain weight,greater than about 200 mg/kg brain weight, greater than about 250 mg/kgbrain weight, greater than about 300 mg/kg brain weight, greater thanabout 350 mg/kg brain weight, greater than about 400 mg/kg brain weight,greater than about 450 mg/kg brain weight, greater than about 500 mg/kgbrain weight.

In some embodiments, the therapeutically effective dose may also bedefined by mg/kg body weight. As one skilled in the art wouldappreciate, the brain weights and body weights can be correlated.Dekaban A S, “Changes in brain weights during the span of human life:relation of brain weights to body heights and body weights,” Ann Neurol1978; 4:345-56. Thus, in some embodiments, the dosages can be converted,as shown in Table 6.

TABLE 6 Correlation between Brain Weights, body weights and ages ofmales Age (year) Brain weight (kg) Body weight (kg) 3 (31-43 months)1.27 15.55 4-5 1.30 19.46

In some embodiments, the therapeutically effective dose may also bedefined by mg/15 cc of CSF. As one skilled in the art would appreciate,therapeutically effective doses based on brain weights and body weightscan be converted to mg/15 cc of CSF. For example, the volume of CSF inadult humans is approximately 150 mL (Johanson C E, et at “Multiplicityof cerebrospinal fluid functions: New challenges in health and disease,”Cerebrospinal Fluid Res. 2008 May 14; 5; 10). Therefore single doseinjections of 0.1 mg to 50 mg protein to adults would be approximately0.01 mg/15 cc of CSF (0.1 mg) to 5.0 mg/15 cc of CSF (50 mg) doses inadults.

It is to be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the enzyme replacement therapy andthat dosage ranges set forth herein are exemplary only and are notintended to hunt the scope or practice of the claimed invention.

Kits

The present invention further provides kits or other articles ofmanufacture which contains the formulation of the present invention andprovides instructions for its reconstitution (if lyophilized) and/oruse. Kits or other articles of manufacture may include container, anIDDD, a catheter and any other articles, devices or equipment useful ininterthecal administration and associated surgery. Suitable containersinclude, for example, bottles, vials, syringes (e.g., pre-filledsyringes), ampules, cartridges, reservoirs, or lyo-jects. The containermay be formed from a variety of materials such as glass or plastic. Insome embodiments, a container is a pre-filled syringe. Suitablepre-filled syringes include, but are not limited to, borosilicate glasssyringes with baked silicone coating, borosilicate glass syringes withsprayed silicone, or plastic resin syringes without silicone.

Typically, the container may holds formulations and a label on, orassociated with, the container that may indicate directions forreconstitution and/or use. For example, the label may indicate that theformulation is reconstituted to protein concentrations as describedabove. The label may further indicate that the formulation is useful orintended for, for example, IT administration. In some embodiments, acontainer may contain a single dose of a stable formulation containing atherapeutic agent (e.g., a replacement enzyme). In various embodiments,a single dose of the stable formulation is present in a volume of lessthan about 15 ml, 10 ml, 5.0 ml, 4.0 ml, 3.5 ml, 3.0 ml, 2.5 ml, 2.0 ml,1.5 ml, 1.0 ml, or 0.5 ml. Alternatively, a container holding theformulation may be a multi-use vial, which allows for repeatadministrations (e.g., from 2-6 administrations) of the formulation.Kits or other articles of manufacture may further include a secondcontainer comprising a suitable diluent (e.g., BWFI, saline, bufferedsaline). Upon mixing of the diluent and the formulation, the finalprotein concentration in the reconstituted formulation will generally beat least 1 mg/ml (e.g., at least 5 mg/ml, at least 10 mg/ml, at least 25mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml). Kitsor other articles of manufacture may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, IDDDs, catheters, syringes, andpackage inserts with instructions for use.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature citations are incorporated byreference.

EXAMPLES Example 1 Evaluation of the Stability of Poloxamer Formulations

The purpose of this example was to evaluate the use of poloxamer instabilizing recombinant human ASA and reducing the level of enzymeprecipitation and/or aggregation. Three separate studies were performed,using different concentrations of a poloxamer in a saline or bufferbased formulation, to examine its role in promoting enzyme stability.Each study were designed to simulate the agitation, pressure andfluctuation in temperature experienced by a protein during commercialmanufacturing. Therefore, it will be appreciate by one skilled in theart, that the results presented in this example represent the effect ofpoloxamers on protein stability during the manufacture, purification,storage and/or re-formulation of a commercial product.

Agitation Studies

This study demonstrates that including a poloxamer in a saline or bufferbased formulation significantly reduces enzyme precipitation includingparticulate (e.g., fibers and flakes, formation. For the study, a seriesof agitation experiments were designed to assess the aggregation ofrecombinantly-prepared human Arylsulfatase A (rhASA) in formulationscontaining various amounts of a poloxamer surfactant, P188, as comparedto a control formulation with no surfactant. To achieve this, rhASA wasprepared and formulated with 0.05% P188, 0.1% P188 or 0.15% P188. TherhASA formulations were agitated by shaking at room temperature for 48hours.

The no-surfactant control developed a few fibers and flakes after 2 hrsof shaking. The number of the particulates increased over time, as theshaking study progressed to 48 hrs time-point (see FIG. 1). Formulationscontaining 0.05% P188 also developed small flakes after 2 hrs ofshaking. After 6 hrs, fibers were also observed. The appearance of thesolution worsened as the shaking was continued to 48 hrs (FIG. 1). Incontrast, the samples containing 0.15% P188 did not develop any fibers,flakes or particular matter over the entire 48 hour period (FIG. 1).This data indicates the use of poloxamer P188 at a concentration closeto 0.15% can increase stability and reduce the level of rhASAprecipitation, following agitation at room temperature. Therefore, forthe next study Poloxamer concentrations of 0.1% and 0.15% were selected.

Pumping and Shaking Studies

For the next study, the objective was to examine the effect of pumpingfollowed by shaking on rhASA formulations in the presence of 0.1% and0.15% of poloxamer 188. The process of high speed pumping followed byshaking is used to serve as an exaggeration of the rigors endured by asample during a routine manufacturing process. In these experiments,0.1% P188 or 0.15% P188 rhASA samples were first pumped through an IVECpump (200 strokes a 700 rpm) and then shaken for up to 48 hours.Immediately after pumping, only a few white fibers were observed for theno-surfactant control samples. However, after pumping and shaking for ˜6hrs, more fibers as well as some particulate matter was observed withinthe no-surfactant control sample. As shown in FIG. 2, after 48 hrs ofshaking of each pumped sample, the worse appearance belonged to thesample with no surfactant followed by the samples (n=3) with 0.1% P188.However, as can be seen in FIG. 3, the samples containing 0.15% P188contained only a few fibers after shaking the pumped material for 48hrs. At all time-points, the pumped but not shaken controls lookedbetter than the pumped and shaken samples, regardless of the presence ofthe surfactant.

Therefore, 0.15% P188 clearly demonstrated protection for the rhASAprotein against stressful conditions such as pumping and agitation,and/or combination of both. 0.1% may still be considered a viable optionfor the long term storage of rhASA at 2-8° C. under less stressfulconditions.

Loop Term Stability Studies

Experiments were conducted to evaluate the effect of poloxamer on thelong term stability of rhASA with 0.1% and 0.15% P188. Specifically, thefollowing two formulations were used: 154 mM NaCl, with 0.1% poloxamer188, at pH 6.0; and 154 mM NaCl, with 0.15% poloxamer 188, at pH 6.0.The samples were placed in long term storage at a temperature of 2-8° C.for a period of 12 months. The samples were analyzed periodically forstability, by evaluating for the presence of precipitate or flocculentmaterial, along with other analytical techniques including, but notlimited to, SEC and SDS-PAGE. Based on the study results (data notshown), the data suggests that rhASA is able to remain soluble andstable at 2-8° C. for at least as period of 12 months, for both the 0.1%and 0.15% poloxamer formulation. Based on the results to-date, the datasuggests that rhASA may be able to remain soluble and stable at 2-8° C.for a period greater than 12 months, for both poloxamer formulations.

In addition, the stability of long term storage of rhASA at 25° C. wasalso evaluated. Specifically, the above two formulations were placed ata temperature of 25° C. for a period of 6 months. The samples wereanalyzed periodically for stability, by evaluating for the presence ofprecipitate or flocculent material, and other analytical techniques(e.g., SEC and SDS-PAGE). The data (not shown) suggests that rhASA isable to remain soluble and stable at 25° C. for a period of 3 months,for both the 0.1% and 0.15% poloxamer formulation.

Therefore, the stability data demonstrate that P188 (e.g., at both 0.1%and 0.15%) facilitates rhASA long term stability.

Example 2 Lyophilization of Poloxamer-Contain rhASA Formulation

In this example, rhASA was lyophilized in the presence of poloxamer as asurfactant in saline-based formulations. The experiment was designed toevaluate the protective property of poloxamer towards rhASA informulations pre and post lyophilized conditions. rhASA samples wereprepared and formulated by dialysis using a 3K MWCO membrane in 110 mMsodium chloride solution. Post dialysis, poloxamer, sucrose, and salinewere added to achieve a final formulation composition containing 25mg/mL rhASA, 110 mM NaCl, 3% sucrose, 0.15% P188, at pH 6.0.

For lyophilization 2 mL of the formulated rhASA sample were filled in 5cc glass vials, partially stoppered with lyophilization stoppers andplaced on a pre-cooled lyophilizer shelf at 5° C. For the initialthermal treatment, the shelf was cooled down to 50° C. at a ramp rate of0.25° C. per minute and held at the set temperature for 120 minutes. Theshelf was then warmed to −30° C. at a ramp rate of 0.25° C. per minuteand held for 360 minutes. The shelf was subsequently re-cooled to −50°C. at a ramp rate of 0.25° C. per minute and held at the set temperaturefor 120 minutes. During primary drying, the shelf was warmed to −34° C.at a ramp rate of 0.25° C. per minute and the chamber pressure wasreduced to 0.133 mbar and held for 6,600 minutes. During secondarydrying, the shelf was warmed to +25° C. at a ramp rate of 0.25° C. perminute and the chamber pressure was reduced to 0.133 mbar and held for420 minutes. Once the lyophilization process has been completed, thevials were back-filled with nitrogen, fully stoppered and sealed with analuminum cap.

Post lyophilization, the appearance was a white solid cake. The cakereconstitution time with water was 10 seconds. The reconstitutedsolution appearance determined, by visual observation was slightlyopalescent with no particles similar to the pre-lyophilize sample. Thepre and post lyophilization pH measurements were 5.94 and 6.00,respectively. The pre and post lyophilization osmolality measurementswere 329 and 321 mOsm/kg, respectively. The percentage main peak asmeasured by size exclusion chromatography for both pre and postlyophilization samples was 99%. The percentage main peak as measured byreserve-phase chromatography for both pre and post lyophilizationsamples was 100%. Protein concentration measurements by absorbance at280 nm for pre and post lyophilization samples were 24.1 and 24.8 mg/mL,respectively. The protein sample attributes did not change pre and postlyophilization as determined by visual observation, pH, osmolality, sizeexclusion chromatography, reversed-phase chromatography, and proteinconcentration.

These data indicates that poloxamer-containing formulations preserverhASA protein stability during the lyophilization process.

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds of theinvention and are not intended to limit the same.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product, or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed, from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising, particularelements, features, etc., certain embodiments of the invention oraspects of invention consist, or consist essentially of, such elements,features, etc. For purposes of simplicity those embodiments have not inevery case been specifically set forth in so many words herein. Itshould also be understood that any embodiment or aspect of the inventioncan be explicitly excluded from the claims, regardless of whether thespecific exclusion is recited in the specification. The publications,websites and other reference materials referenced herein to describe thebackground of the invention and to provide additional detail regardingits practice are hereby incorporated by reference.

1-47. (canceled)
 48. A stable formulation for intrathecal administrationcomprising an arylsulfatase A (ASA) protein and a poloxamer, wherein theASA protein is present at a concentration of at least approximately 0.1mg/ml, and wherein less than 5% of the arylsulfatase A (ASA) proteinexists in aggregated form.
 49. The stable formulation of claim 48,wherein the poloxamer is selected from the group consisting ofPoloxamers 101, 105, 108, 122, 123, 124, 181, 182, 183, 184, 185, 188,212, 215, 217, 231, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334,335, 338, 401, 402, 403, 407, and combination thereof.
 50. The stableformulation of claim 49, wherein the poloxamer is poloxamer
 188. 51. Thestable formulation of claim 48, wherein the poloxamer is present at aconcentration ranging from approximately 0.05-0.50%, ranging fromapproximately 0.05-0.20%, of approximately 0.15%, or of approximately0.1%.
 52. The stable formulation of claim 48, wherein less than 4%, lessthan 3%, less than 2%, or less than 1% of the arylsulfatase A (ASA)protein exists in aggregated form.
 53. The stable formulation of claim48, wherein the ASA protein is present at a concentration selected fromabout 1 mg/ml, 10 mg/ml, 30 mg/ml, 50 mg/ml, or 100 mg/ml.
 54. Thestable formulation of claim 48, wherein the ASA protein comprises anamino acid sequence having at least 80% identity to SEQ ID NO:1 or theASA protein comprises the amino acid sequence of SEQ ID NO:1.
 55. Thestable formulation of claim 48, wherein the ASA protein is produced froma human cell line or from CHO cells.
 56. The stable formulation of claim48, wherein the formulation further comprises salt.
 57. The stableformulation of claim 56, wherein the salt is NaCl and is present at aconcentration ranging from approximately 0-300 mM, ranging fromapproximately 80-160 mM, or of approximately 154 mM.
 58. The stableformulation of claim 48, wherein the stable formulation furthercomprises a buffering agent selected from the group consisting ofphosphate, acetate, histidine, succinate, citrate, Tris, andcombinations thereof.
 59. The stable formulation of claim 58, whereinthe buffering agent is phosphate.
 60. The stable formulation of claim48, wherein the formulation has a pH of approximately 3-8.0,approximately 6.0-6.5, or approximately 6.0.
 61. The stable formulationof claim 48, wherein the formulation is a liquid formulation or isformulated as lyophilized dry powder.
 62. The stable formulation ofclaim 48, wherein the formulation further comprises a stabilizing agentselected from the group consisting of sucrose, glucose, mannitol,sorbitol, polyethylene glycol (PEG), histidine, arginine, lysine,phospholipids, trehalose and combination thereof.
 63. A stableformulation for intrathecal administration comprising an arylsulfatase A(ASA) protein at a concentration ranging from approximately 0.1-100mg/ml, a poloxamer at a concentration ranging from approximately0.05-0.50%, NaCl at a concentration ranging from approximately 0-300 mM,and a pH of 3-8.0, wherein less than 5% of the ASA protein exists inaggregated form.
 64. A container comprising a single dosage form of astable formulation according to claim
 48. 65. The container of claim 64,wherein the container is selected from an ampule, a vial, a cartridge, areservoir, a lyo-ject, or a pre-filled syringe.
 66. The container ofclaim 64, wherein the stable formulation is present in a volume of lessthan about 50.0 ml or less than about 5.0 ml.
 67. A method of treatingmetachromatic leukodystrophy (MLD) disease comprising a step ofadministering intrathecally to a subject in need of treatment aformulation comprising an arylsulfatase A (ASA) protein at aconcentration ranging from approximately 0.1-100 mg/ml, a poloxamer at aconcentration ranging from approximately 0.05-0.50%, NaCl at aconcentration ranging from approximately 0-300 mM, and a pH of 3-8.0,wherein less than 5% of the ASA protein exists in aggregated form.